3.8 Control of Gene Expression Flashcards
Define gene mutation
change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide
When are errors in DNA most likely to occur?
DNA replication
What are the main types of gene mutations
Addition
-insertion of a base
-causes a frameshift to the right
-all subsequent codons are altered
-hence all subsequent amino acids may differ
-results in a different polypeptide being produced
Deletion
-removal of a base
-causes a frameshift to the left
-all subsequent codons are altered
-hence all subsequent amino acids may differ
-results in a different polypeptide being produced
Substitution
-a base in the DNA sequence is randomly swapped for a different base
-only change the amino acid for the triplet (group of three bases) in which the mutation occurs; it will not have a knock-on effect
-can take three forms:
-silent mutations – doesn’t alter amino acid sequence of the polypeptide as the genetic code is degenerate
-missense mutations – alters a single amino acid in the polypeptide chain (e.g. sickle cell anaemia)
-nonsense mutations – creates a premature stop codon = polypeptide chain produced to be incomplete - affects the final protein structure and function
Inversion
-usually occurs during crossing-over in meiosis
-DNA of a single gene is cut in two places
-cut portion is inverted 180° then rejoined to the same place within the gene
-leads to a large section of the gene being ‘backwards
-hence multiple amino acids are affected
-frequently result in a non-functional protein
-often harmful as the original gene can no longer be expressed from that chromosome
-effect may be lessened if the other chromosome in the pair carries a working gene
Duplication
-whole gene or section of a gene is duplicated so that two copies of the gene/section appear on the same chromosome
-original version of the gene remains intact hence not harmful
-overtime, the second copy can undergo mutations which enable it to develop new functions
-important source of evolutionary change
-alpha, beta and gamma haemoglobin genes evolved due to duplication mutations
Translocation
-gene is cut in two places
-section of the gene that is cut off attaches to a separate gene
-hence the cut gene is now non-functional due to having a section missing
-the gene that has gained the translocated section is likely to also be non-functional
-if section of a proto-oncogene is translocated onto a gene controlling cell division, it could boost expression and lead to tumours
-similarly, if a section of a tumour suppressor gene is translocated and the result is a faulty tumour suppressor gene, this could lead to the cell continuing replication when it contains faulty DNA
sme lesson 2
The Effect of Genetic Mutations
Causes of mutations
-exposure to mutagenic agents can increase the rate of mutation, they include
-high energy ionising radiation, such as alpha, beta or gamma radiation
chemicals, such as nitrogen dioxide or benzopyrene from tobacco smoke
The effect of gene mutations on phenotype
-proteins affect the phenotype of an organism via specific cellular mechanisms
-e.g., a mutation in the TYR gene in humans affects the structure of an enzyme that is needed for the production of the pigment melanin
-phenotype of the human is affected by the lack of melanin
-individuals with the mutation have albinism; very pale skin and hair
how transcription factors work
Transcription Factors
-protein that controls the transcription of genes by binding to a specific region of DNA
-eukaryotes use transcription factors to control gene expression
-estimated that ~10% of human genes code for transcription factors
-allow organisms to respond to their environment
The structure of a gene
‘Upstream’ refers to the DNA before the start of the coding region
-promoter = section of DNA upstream of the coding region that is the binding site for proteins that control the expression of the gene, including:
-RNA polymerase
-transcription factors
While DNA is translated in the 3’ to 5’ direction, it is transcribed in the 5’ to 3’ direction to produce messenger RNA (mRNA)
How transcription factors work
-enter the nucleus from the cytoplasm through nuclear pores
-activated through a signalling pathway that usually starts from outside the cell
Some transcription factors bind to the promoter region of a gene
This binding can either allow or prevent the transcription of the gene from taking place
Transcription factors interact with RNA polymerase, either by assisting RNA polymerase binding to the gene (to stimulate expression of the gene) or by preventing it from binding (to inhibit gene expression)
Therefore, the presence of a transcription factor will either increase or decrease the rate of transcription of a gene
For example, oestrogen is a hormone, found in mammals, that works as a transcription factor alongside specific proteins that activates the transcription of many genes
spare 2
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oestrogen as a TF
-steroid hormone = small, hydrophobic, lipid-based
-can diffuse through the cell membrane + directly into nucleus through nuclear pores
-involved in controlling the female fertility cycle
-responsible for stimulating sperm production in males
The oestrogen stimulation pathway:
-oestrogen diffuses through cell surface membrane into cytoplasm
-diffuses through a nuclear pore into the nucleus
-within the nucleus, oestrogen attaches to an ER alpha oestrogen receptor that is held within a protein complex
-causes the ERα oestrogen receptor to undergo a conformational change
-new shape of the ERα oestrogen receptor allows it to detach from the protein complex and diffuse towards the gene to be expressed
-ERα oestrogen receptor binds to a cofactor
-enables it to bind to the promoter region of the gene
-stimulates RNA polymerase to bind
-gene can now be transcribed
Define stem cells
-cell that can divide (by mitosis) an unlimited number of times
-can remain as stem cell or develop into a specialised cell by differentiation
-capacity for self-renewal
What is small interfering RNA?
-small, double-stranded RNA
-RNAi inhibits translation of the mRNA produced from target genes in eukaryotes and some prokaryotes
-siRNA bind to mRNA that has been transcribed from target genes (the genes to be ‘silenced’)
-due to complementary base sequence
-each siRNA is attached to a protein complex which is able to breakdown the mRNA that has been transcribed from target genes
-hence, mRNA is unable to be translated into proteins
RNA interference pathway
-RNAi is a form of post-transcriptional modification
-sequence-specific silencing of gene expression
-occurs in the cytoplasm
-double stranded RNA (dsRNA) is produced by RNA-dependent RNA polymerases (RDRs)
-dsRNA is hydrolysed by enzyme (dicer) into smaller fragments called siRNA (around 23 nucleotides long)
-in the cytoplasm, siRNAs bind to protein complexes (uses energy from ATP to separate both strands of the siRNA)
-exposes nucleotide bases
-target mRNA leaves the nucleus and enters the cytoplasm
-single-stranded siRNA binds to the target mRNA through complementary base pairing
-mRNA molecule is cut into fragments by the enzyme/protein complex associated with the siRNA
-cut mRNA cannot be translated
-cannot produce proteins
-fragmented mRNA is broken down into RNA nucleotides by enzymes
Therapeutic Application of RNA interference
-siRNAs created against viral genetic material will signal for their degradation
-stops the virus from using the host’s cellular machinery to replicate itself
-can be used in cancer treatment by targeting oncogenes that have been expressed or upregulated
-reduces the number of proteins produced that can lead to or maintain cancerous growth
Define potency
What are totipotent cells?
potency = ability of stem cells to differentiate into more specialised cell types
-can divide and produce any type of body cell
-aka ‘embryonic stem cells’
-exist for a limited time in early mammalian embryos, as well as in extra-embryonic cells
-the zygote formed when a sperm cell fertilises an egg cell is totipotent
-initially, totipotent cells in the embryo are unspecialised
-during development, totipotent cells begin to translate only part of their DNA
-results in cell specialisation
-specialised cells then form tissues and are no longer classed as totipotent
Pluripotent stem cells
-embryonic stem cells that can differentiate into any cell type found in an embryo
-unable to differentiate into extra-embryonic cells (cells that make up the placenta)
-can divide in unlimited numbers and keep replacing themselves
Induced pluripotent stem cells (iPS cells)
-can be produced from adult somatic cells using appropriate transcription factors
-these cause specific genes to be expressed which dedifferentiate a cell back to its pluripotent state
-each individual can have their own pluripotent stem cell line produced from their body’s cells
-could potentially be used to generate transplants without the risk of immune rejection
multipotent stem cells
-cells must differentiate and specialise to fulfil particular roles
-hence, these adult cells gradually lose their ability to divide
-but adult stem cells remain to produce new cells for essential processes (growth, cell replacement and tissue repair)
-these can divide (by mitosis) an unlimited number of times
-only able to produce a limited range of cell types
-e.g. stem cells found in bone marrow are multipotent adult stem cells
-can only differentiate into blood cells (red blood cells, monocytes, neutrophils and lymphocytes)
-in adults, stem cells can be found throughout the body (eg. in the bone marrow, skin, gut, heart and brain)
-research is being carried out on stem cell therapy
-introduction of adult stem cells into damaged tissue to treat diseases (eg. leukemia) and injuries (eg. skin burns)
What are unipotent cells?
-adult cells that can only differentiate into one type of cell
-e.g. heart muscle cells (cardiomyocytes) can generate new cardiomyocytes through the cell cycle to build and replace heart muscle
-most cells in animal bodies are unipotent
use of embryonic stem cells
-ability to differentiate into multiple cell types = huge potential in the therapeutic treatment of disease and producing transplants
-embryonic cells can be:
-totipotent if taken in the first 3-4 days after fertilisation
-pluripotent if taken on day 5
-embryos used for research are fertilised in vitro and specifically donated for this purpose
use of multipotent adult cells
Adult stem cells can divide (by mitosis) an unlimited number of times but they are only able to produce a limited range of cell types
A small number of adult stem cells are found in certain tissues within the body such as:
Bone marrow - used to produce different types of blood cell
Brain - used to produce different types of neural and glial cells
However, small numbers of stem cells (known as adult stem cells) remain to produce new cells for the essential processes of growth, cell replacement and tissue repair
Research is being carried out on stem cell therapy, which is the introduction of adult stem cells into damaged tissue to treat diseases (eg. leukaemia) and injuries (eg. skin burns)
The use of adult stem cells is less controversial than embryonic stem cells because the donor is able to give permission
For example, many people donate bone marrow to help treat leukaemia patients
However, if multipotent stem cells are being donated from one person to another they need to be a close match in terms of blood type and other body antigens
There is a chance that the cells used are rejected by the patient’s immune system
use of induced pluripotent stem cells
iPS cells have been developed by scientists using an adult’s somatic cells that are unipotent (fully differentiated)
As all somatic cells contain the same genetic material scientists are able to use specific transcription factors to target the genes that control pluripotency
Scientists ‘switch on’ these genes that are usually silenced in differentiated cells which allows them to revert back to pluripotent cells
The resultant pluripotent cells can then be used to produce any type of cell required for repair/treatment of the body
iPS cells could therefore be used instead of embryonic cells
This would avoid the ethical issues associated with using embryonic stem cells
However, this technique is not verified yet as during research iPS cells have caused tumour formation
This is thought to be because some of the genes switched on will control the cell cycle and its regulation, which if uncontrolled will lead to tumour formation
beneftits social issues etc table from sme
intro to epigenetics
-change in gene function without changing the DNA base sequence
-epigenome: all of the chemical modifications to all histone proteins and DNA (except base changes) in an organism
-in eukaryotic cells, nuclear DNA is wrapped around proteins called histones
-identical twins become more distinguishable with age despite having identical DNA
-due to their epigenomes change independently
-these changes are caused by environmental changes
-e.g. smoking, stress, exercise and diet
-internal signalling from the body’s own cells can also cause modifications to occur
-chemical modification of histones and DNA changes intermolecular bonding between histones and DNA
-hence controls how tightly the DNA is wound around them
When DNA is wound more tightly in a certain area…
-gene and promoter regions are more hidden from transcription factors and RNA polymerase
-the genes on these section of DNA are ‘switched off’
-modification of histones is reversible
-hence can vary with age and in different cell types
acetylation of histones
-acetyl groups (COCH3) can be added to lysine amino acids on histone proteins
-decreases the positive charge on histone proteins
-decreases attraction to phosphate groups on DNA
-causes chromatin to decondense
-DNA is less tightly wrapped around histones
-hence transcription factors and RNA polymerase can bind to DNA more easily
-hence gene expression is stimulated
-deacetylation returns lysine to its positively charged state
-has a stronger attraction to the DNA molecule
-hence inhibits transcription
methylation of DNA
-methyl groups (CH3) can be added to cytosine bases
-methylated bases attract proteins that bind to the DNA
-inhibits transcription of affected gene
Define epigenetic imprinting
Inheritance of epigenetic modifications
Give an example of a condition with epigenetic links
Epigenetic imprinting: DNA methylation of certain genes
-occurs during the formation of oocytes and sperm cells
-child inherits two sets of DNA, one from each parent
-each has its own epigenetic imprint
-imprinting is reversible, so the maternal epigenetic imprints that are inherited by a male will become paternal imprints when his sperm are produced
-hence he passes on paternally imprinted DNA
-Prader-Willi syndrome is an autosomal dominant condition
-has epigenetic links
-caused by the silencing of an allele on chromosome 15
-severity of the syndrome depends on whether an individual receives the affected DNA from a parent
-if the mother is a carrier for the defective chromosome, individuals that inherit the chromosome do not develop the syndrome
-however, if the defective chromosome is inherited from the father, the individual will develop the syndrome
-leads to loss of function of some genes
Epigenetic cancer treatment
what is an oncogene?
what is an tumour suppressor gene?
-DNA in human tumour cells have changes in DNA methylation and histone acetylation
-causes tumour suppressor genes to be silenced and oncogenes to be activated
-leads to deregulation of the cell cycle + formation of tumours
-some cancer treatments involve drugs that reverse the epigenetic changes through the removal of acetyl and methyl tags
Removal of methyl groups from the DNA of tumour suppressor genes
-genes can be expressed
-proteins produced can then regulate the cell cycle
-stops tumours forming from faulty or cancerous cells
Removal of acetyl groups from histone proteins attached to oncogenes
-causes the DNA to wrap more tightly
-silences these genes
-reduced expression of oncogenes stops cancer
-as faulty cells are able to die through programmed cell death (apoptosis) rather than continuing to replicate, causing cancer
Oncogene:
-mutated version of a proto-oncogene that controls cell division
-causes cells to divide too quick, leading to tumour formation
Tumour suppressor gene:
-gene which controls cell division by slowing it down
what are tumours
-groups of abnormal cells that form lumps or growths
-different characteristics depending on whether they are malignant or benign
All tumours may cause harm to the body by:
-damaging the organ in which the tumour is located
-causing blockages or obstructions
-damaging other organs by exerting pressure
malignant tumours
-cancerous and grow rapidly
-invade and destroy surrounding tissues
-cells within malignant tumours secret chemicals
-cause formation of blood vessels to supply the tumour with nutrients, growth factors and oxygen
-cells can break off these tumours and spread to other parts of the body through the bloodstream or lymphatic system (metastasis)
-may affect multiple organs
-when removed through surgery, malignant tumours can still grow back
The formation of malignant tumours can be initiated by carcinogens such as:
-UV or X-ray exposure
-tobacco from cigarettes
-asbestos
-processed meat
benign tumours
-not cancerous and grow slowly
-no invasion of other tissues = no metastasis
-benign tumours can cause damage, e.g. blockages, exerting pressure on the organ
-when removed, they do not usually grow back
Formation of benign tumours can be initiated by:
-inflammation or infection
-injury
-diet
-genetics
-toxins and radiation
Examples of benign tumours are:
-polyps found in the nose, colon and ovaries
-non-cancerous brain tumours
-warts, caused by a viral infection
how do tumours develop
-cancerous cells divide repeatedly and uncontrollably
-starts when expression of genes that control cell division changes
-if the mutated gene is one that causes cancer it is referred to as an oncogene
-most mutations result in early cell death or destruction by the body’s immune system
-usually not harmful as most cells can easily be replaced
Tumour suppressor genes
Tumour suppressor genes are normal genes that code for proteins that regulate the cell cycle
The proteins encoded for by tumour suppressor genes carry out the following functions:
DNA repair
Slowing the cell cycle by ensuring checks are made
Signalling apoptosis (cell death) when the cell is faulty
These proteins ensure that cells do not replicate if they contain mutated DNA or are faulty as these characteristics can lead to tumour formation
Tumours develop if tumour suppressor genes are mutated or silenced
Silencing can occur through epigenetic changes and RNAi
Hypermethylation of DNA (over-addition of methyl groups to cytosine nucleotides) causes transcription-inhibiting proteins to bind the DNA, if this occurs around tumour suppressor genes this could result in tumour development as the necessary regulatory proteins coded for by tumour suppressor genes will not be produced
RNA interference by siRNAs targeting tumour suppressor genes for breakdown can also lead to tumour development for the same reason
Oncogenes
Proto-oncogene: normal gene that codes for proteins that regulate cell growth (growth factors) and cell differentiation
can mutate to become oncogenes
-cause of mutations = carcinogens (e.g. exposure to UV, X-rays and smoking)
-oncogenes have the ability to cause cancer through unregulated cell growth
-mutations that produce oncogenes usually causes the proteins that stimulate cell growth and division to be constantly activated
-activation of these proteins speeds up the cell cycle
Mutation of proteins involved in apoptosis can lead to tumour formation
-mutations of proto-oncogenes to produce oncogenes can occur through inversion or translocation mutations
-an activating segment of a gene is attached to a proto-oncogene
-leads to gene expression being upregulated
-or protein produced being constantly activated
-unable to be switched off
Common ways of activating/deactivating proteins:
-phosphorylation - phosphate group from ATP is added
-complex formation - binding to another protein or coenzyme = change in protein’s conformation, potentially opening up another binding site or revealing an active site
Oestrogen-dependent breast tumours
-steroid hormone that upregulates transcription of certain genes through the stimulation of the ERα oestrogen receptor
-high concentrations of oestrogen can lead to the development of breast cancer
-high concentrations can be due to over-expression of the oestrogen gene or from supplementary oestrogen taken in medication
-about 70% of breast tumours are categorised as oestrogen receptor-positive
-oestrogen is needed by these tumours to stimulate the expression of cell cycle genes that lead to growth and replication
-oestrogen receptors in cancer cells promote cell growth when stimulated by oestrogen
-the genes are switched on through the oestrogen dependent gene expression pathway (oestrogen diffuses into the cell and through a nuclear pore until it reaches the oestrogen receptors)
-main treatment = drug called tamoxifen
-has a similar chemical structure to oestrogen
-therefore acts as a competitive inhibitor
-tamoxifen permanently binds to oestrogen receptors
-stops oestrogen from binding
-inhibits the receptor’s action
-hence the genes that are usually upregulated by oestrogen are not expressed
-due to oestrogen receptor not being able to bind to the promoter
-tumours can’t grow
define the following:
Benign
Malignant
Metastasis
Oncogene
Proto-oncogene
Tumour suppressor genes
- Benign—tumors that stay in one place and do not affect
surrounding tissue - Malignant—tumors that invade surrounding tissue
- Metastasis—cells from original tumor break away and
start new cancers somewhere else—metastasize. Cells
break off from tumor, enter bloodstream, move through
blood vessel wall and invade tissue - Tumor suppressors–another class of genes involved in
cancer - Carry instructions for producing proteins that suppress
cell division if conditions are unfavorable - Proto-oncogenes–genes that provide instructions for
proteins that regulate the cell cycle - Oncogenes—mutated proto-oncogenes
differences between cancer and normal
Cell cultures of these normal cells can live and divide in glassware, but cease cell division once the space has been used up, for example the Petri dish or glass bottle surface has been covered by actively dividing cells
cancer cells do not stop dividing and, unlike healthy tissue, they do not form ordered layers, instead they form heaps of disordered cells
Describe how the process of cell division can lead to mutations and cancer
Explain how Breast cancer results from mutations in the BRACA1 and BRACA2 tumour suppressor genes.
-BRCA-1 = example of a human tumour suppressor gene
-expressed mainly in breast tissue
-role of the BRCA-1 protein: repair broken or mutated DNA
-if the BRCA-1 protein cannot repair the DNA, it signals for apoptosis to begin
-in breast cancers, BRCA-1 expression is inhibited
-leads to a lack of DNA repair and apoptosis
-hence cancerous tumours form
the role of miRNAs in the control of cell proliferation.
protein called exportin-5 transports a hairpin primary MiRNA out of the nucleus
add more from pic ppt
-action of the dicer enzyme which splits off the loop of the miRNA, which then leaves one of the strands to join to an Argonaute protein
-The remaining strand is discarded. The Argonaute protein offers the miRNA fragment to the messenger RNA.
Plant cells and animal cell differ in their handling of the single stand of miRNA, in both the miRNA combines with mRNA but base pairing mismatches occur between nucleotides in the case of animal cells whereas mismatches do not
happen in plant cells
-net result in both cases is the destruction of the mRNA strand
-therefore termination of its ability to translate the production of protein at the Ribosome
Human Genome Project
In the 1980s Cambridge scientists had been working on sequencing the genome of a nematode. As they progressed they realised that the technology used in this research could be applied to the human genome
The Human Genome Project (HGP) began in 1990 as an international, collaborative research programme
It was publicly funded so that there would be no commercial interests or influence
DNA samples were taken from multiple people around the world, sequenced and used to create a reference genome
Laboratories around the globe were responsible for sequencing different sections of specific chromosomes
It was decided that the data created from the project would be made publicly available
As a result, the data can be shared rapidly between researchers
The information discovered could also be used by any researcher and so maximised for human benefit
By 2003 the human genome had been sequenced to 99.9% accuracy
The finished genome was over 3 billion base pairs long but contained only about 25,000 genes
This was much less than expected
Following the success of sequencing the human genome scientists have now moved onto sequencing the human proteome
The proteome is all of the proteins that can be produced by a cell
Although there are roughly 25,000 genes within the genome there are many more proteins within the proteome. This may is due to processes such as alternative splicing and post-translational modification
There is also work being done on the human epigenome
These are the inherited changes in DNA that do not involve a change in DNA base sequenc
Applications of human genome project
The information generated from the HGP has been used to tackle human health issues with the end goal of finding cures for diseases
Scientists have noticed a correlation between changes in specific genes and the likelihood of developing certain inherited diseases
The mechanism which causes these inherited diseases to develop is not yet understood. It is being actively researched by thousands of scientists
For example, several genes within the human genome have been linked to increased risk of certain cancers
If an individuals BRCA1 and BRCA2 genes are mutated then they are substantially more likely to develop breast cancer
There have also been specific genes linked to the development of Alzheimer’s disease
Sequencing DNA to Determine Protein Sequences
The genome of simpler organisms can be used to obtain the proteome (the sequences of the proteins) of the organism
This information can be used for a range of applications
For example, identifying potential antigens for use in vaccine production
Large databases are created containing information about an organism’s gene sequences and amino acid/protein sequences
Once a genome is sequenced bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available. This can help to find the degree of similarity between organisms which then gives an indication of how closely related the organisms are and whether there are organisms that could be used in experiments as a model for humans (eg. the fruit fly Drosophila)
The nematode Caenorhabditis elegans is an animal that has been used as a model organism for studying the genetics of organ development, neurone development and cell death. It was the first multicellular organism to have its genome fully sequenced and as it has few cells (less than 1000) and is transparent. It has been a useful model
Non-Coding DNA & Regulatory Genes
It can be highly difficult to translate the genome of complex organisms into their proteome
Determining the proteome of humans is difficult as large amounts of non-coding DNA are present in human genomes
It can be very hard to identify these sections of DNA from the coding DNA
The presence of regulatory genes and the process of alternative splicing in human genomes also affects gene expression and the synthesis of proteins
The proteome is larger than the genome due to:
Alternative splicing
Post-translational modification of proteins (often takes place in the Golgi apparatus)
Automated DNA sequencing
Automated DNA sequencing makes use of the chain-termination technique
A short length of DNA is chosen and inserted into a vector as a single strand of DNA
A primer is annealed to the start of the recombinant DNA
During the incubation period, DNA polymerase is added to the recombinant DNA alongside a mixture of deoxyribose nucleotides (deoxynucleotides) containing all 4 bases
Usually, DNA polymerase attaches to the primer and begins DNA replication of the single strand recombinant DNA
Hydrogen bonds form between the complementary bases on deoxyribose nucleotides
However, a mixture of dideoxyribose nucleotides (dideoxynucleotides) are also present (containing all 4 bases)
DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of DNA replication
When there is a sufficient ratio of deoxynucleotides to dideoxynucleotides complementary DNA chains of varying lengths are produced
These chains can vary in length from one nucleotide to several hundred nucleotides
Each type of dideoxynucleotide is labelled using a specific fluorescent dye
Dideoxynucleotides with an adenine base (ddNA) are labelled green
Dideoxynucleotides with a thymine base (ddNT) are labelled red
Dideoxynucleotides with a cytosine base (ddNC) are labelled blue
Dideoxynucleotides with a guanine base (ddNG) are labelled yellow
Once the incubation period has ended and the dideoxynucleotides have bound to their complementary bases the DNA chains are removed from the template DNA
The single-stranded DNA chains are then separated according to size using a specific type of electrophoresis that takes place inside a capillary tube
This type of electrophoresis technique has a very high resolution. It is capable of separating chains of DNA that vary by only one nucleotide in length
A laser beam is used to illuminate all of the dideoxynucleotides and a detector then reads the colour and position of each fluorescence
The detector feeds the information into a computer where it is stored or printed out for analysis
An automated DNA sequencing machine can read roughly 100 different DNA sequences within 2 hours
Sequencing Methods
DNA sequencing allows for the base sequence of an organism’s genetic material to be identified and recorded
Sequencing methods are continuously evolving and becoming faster. Advances in technology have allowed scientists to rapidly sequence the genomes of organisms
Most sequencing methods used are now automated
The data obtained from sequencing can be entered into computers with specialised programmes that compare the base sequences of different organisms
DNA sequencing
All methods of DNA sequencing use dideoxyribose nucleotides
A dideoxyribose molecule is very similar in structure to ribose molecules and deoxyribose molecules
It has one less oxygen atom than a deoxyribose molecule and two fewer oxygen atoms than a ribose molecule
Dideoxyribose can form nucleotides in the same way that ribose and deoxyribose molecules do, by binding to a phosphate molecule and an organic base
Dideoxyribose nucleotides can pair with deoxyribose nucleotides on the template strand during DNA replication
They will pair with nucleotides that have a complementary base
When DNA polymerase encounters a dideoxyribose nucleotide on the developing strand it stops replicating. This is the chain-termination technique that is used for DNA sequencing
Interpreting the results from manual DNA sequencing
Manual DNA sequencing follows a similar process to automated sequencing but there are some key differences:
A separate run is required for each type of dideoxynucleotide - ddNA, ddNT, ddNC and ddNG
The dideoxynucleotides are labelled using radioactivity instead of fluorescent dyes
After the incubation period, the four separate mixtures are added to separate wells in a gel and separated using gel electrophoresis
A Southern transfer is made using the electrophoresis gel and an autoradiograph is taken of the Southern transfer
The DNA sequence can be interpreted using the autoradiograph
Below each well there is a track of bands (DNA fragments) produced from DNA replication in the presence of each type of dideoxynucleotide (ddNA, ddNT, ddNC and ddNG)
The band that moves the furthest distance is the smallest DNA fragment
The smallest DNA fragment that can be formed from the chain termination technique is one nucleotide long so whichever track this band is present in determines the first base in the sequence of the developing strand
The second smallest DNA fragment that has travelled the second furthest will determine the second base in the sequence and the third smallest DNA fragment that has travelled the third furthest will determine the third base in the sequence etc.
recombinant dna tech
overview of Recombinant DNA
The genetic code is universal, meaning that almost every organism uses the same four nitrogenous bases – A, T, C & G. There are a few exceptions
The genetic code is the basis for storing instructions that, alongside environmental influences, dictate the behaviour of cells and as a result, the behaviour of the whole organism
The universal nature of the genetic code means that the same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species)
Thus scientists have been able to artificially change an organism’s DNA by combining lengths of nucleotides from different sources (typically the nucleotides are from different species)
The altered DNA, with the introduced nucleotides, is called recombinant DNA (rDNA)
If an organism contains nucleotide sequences from a different species it is called a transgenic organism
Any organism that has introduced genetic material is a genetically modified organism (GMO)
The mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism
what is Recombinant DNA technology
-form of genetic engineering
-involves the transfer of DNA fragments from one organism into another organism
-resulting genetically engineered organism will then contain recombinant DNA and will be a genetically modified organism (GMO)
In order for an organism to be genetically engineered the following steps must be taken:
Identification of the DNA fragment or gene
Isolation of the desired DNA fragment
Multiplication of the DNA fragment (using polymerase chain reaction - PCR)
Transfer into the organism using a vector (e.g. plasmids, viruses, liposomes)
Identification of the cells with the new DNA fragment (by using a marker), which is then cloned
Genetic engineers need the following to modify an organism:
-enzymes (restriction endonucleases, ligase and reverse transcriptase)
-vectors - used to deliver DNA fragments into a cell (eg. plasmids, viruses and liposomes)
-markers - genes that code for identifiable substances that can be tracked
-eg. GFP - green fluorescent protein which fluoresces under UV light or GUS - β-glucuronidase enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent)
Genetic engineering is being used in the new field of science called synthetic biology
This is an area of research that studies the design and construction of different biological pathways, organisms and devices, as well as the redesigning of existing natural biological systems
how are DNA fragments produced
Genetic engineering is the deliberate modification of a specific characteristic (or characteristics) of an organism. The technique involves removing a gene (or genes), with the desired characteristic, from one organism and transferring the gene (using a vector) into another organism where the desired gene is then expressed
The gene with the specific characteristic that is required can be obtained in the following ways:
Extracting the gene from the DNA of a donor organism using enzymes (restriction endonucleases)
Using reverse transcriptase to synthesise a single strand of complementary DNA (cDNA) from the mRNA of a donor organism
Synthesising the gene artificially using nucleotides in a “gene machine”
Extraction of genes
The extraction of the gene (containing the desired nucleotide sequence) from the donor organism occurs using restriction endonucleases
Restriction endonucleases are a class of enzymes found in bacteria. They are used as a defence mechanism by bacteria against bacteriophages (viruses that infect bacteria, also known as phages)
The enzymes restrict a viral infection by cutting the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule. This is why they are called restriction endonuclease (‘endo’ means within)
They are also referred to as restriction enzymes
There are many different restriction endonucleases because they bind to a specific restriction site (specific sequences of bases) on DNA, eg. HindIII will always bind to the base sequence AAGCTT
The restriction endonucleases are named according to the bacteria they are sourced from and which numbered enzyme it is from that source (eg. HindIII comes from Haemophilus influenzae and it is the third enzyme from that bacteria)
Restriction endonucleases will separate the two strands of DNA at the specific base sequence by ‘cutting’ the sugar-phosphate backbone in an uneven way to give sticky ends or straight across to give blunt ends
Sticky ends result in one strand of the DNA fragment being longer than the other strand
The sticky ends make it easier to insert the desired gene into another organism’s DNA as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA that have been cut with the same restriction enzyme
When using genes isolated by restriction endonucleases that give blunt ends nucleotides can be added to create sticky ends
mRNA & reverse transcriptase
Another method to isolate the desired gene is to use the mRNA that was transcribed for that gene
Once isolated, the mRNA is then combined with a reverse transcriptase enzyme and nucleotides to create a single strand of complementary DNA (cDNA)
Reverse transcriptase enzymes are sourced from retroviruses and they catalyse the reaction that reverses transcription. The mRNA is used as a template to make the cDNA
DNA polymerase is then used to convert the single strand of cDNA into a double-stranded DNA molecule which contains the desired code for the gene
This technique for isolating the desired gene is considered advantageous as it is easier for scientists to find the gene because specialised cells will make very specific types of mRNA (eg. β-cells of the pancreas produce many insulin mRNAs) and the mRNA (therefore the cDNA) does not contain introns
Artificial synthesis using a “gene machine”
-scientists can use the knowledge of the genetic code (i.e. which amino acids are required) and computers to generate the nucleotide sequence (rather than an mRNA template) to produce the gene
-short fragments of DNA are first produced
-joined to make longer sequences of nucleotides
-inserted into vectors (eg. plasmids)
-this method is being used to create novel genes contained in vaccines and even to synthesise new bacteria genomes
What is PCR
-in vitro method of DNA amplification
-produces large quantities of specific fragments of DNA or RNA from very small quantities
-involves three key stages per cycle
-in each cycle the DNA is doubled
-thermocycler automatically provides the optimal temperature for each stage and controls the length of time spent at each stage
What is required for PCR?
Target DNA/RNA being amplified
Primers (forward and reverse)
-short sequences of single-stranded DNA
-have base sequences complementary to the 3’ end of the DNA or RNA being copied
-identifies the region that DNA polymerase should start building new strands from
DNA polymerase
-enzyme used to build the new DNA or RNA strand
-Taq polymerase is most commonly used
-comes from a thermophilic bacterium Thermus aquaticus
-hence it does not denature at the high temperature involved during the first stage of the PCR reaction
-its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
Free nucleotides – used in the construction of the DNA or RNA strands
Buffer solution – to provide the optimum pH for the reactions to occur in
Stages of PCR
Acronym: Divas Always Eat
Denaturation
-double-stranded DNA is heated to 95°C
-breaks hydrogen bonds between both DNA strands
Annealing
-temperature is decreased to between 50 - 60°C
-primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
Extension
-temperature is increased to 72°C for at least a minute
-optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules
the polymerase chain reaction (PCR) can be used to produce large quantities of DNA. describe how the PCR is carried out
DNA is heated to 95 degrees Celsius
strands separate
cooled to 55
primers bind
nucleotides attach by complementary base pairing
temp increased to 72
DNA polymerase joins nucleotides together
cycle repeats to form more copies
In Vivo Method: Culture of Transformed Host Cells
-gene cloning can also be carried out in vivo, using bacteria
-they are the most common host cells for this method as they increase in numbers rapidly and are relatively easy to culture
Process:
-a DNA fragment is isolated
-desired gene(s) is obtained by one of three methods
-extraction using restriction endonucleases
-conversion of mRNA to cDNA using reverse transcriptase
-artificial synthesis in a “gene machine”
-promoter and terminator regions are added to DNA fragments to ensure replication
-DNA fragments are inserted into vectors (e.g. plasmid), using restriction endonucleases and ligase enzymes
-vectors are transported into bacterial host cells
-the cells containing the modified plasmids are described as transformed host cells
-bacteria rapidly multiply in number
-marker genes are used to identify the successfully transformed bacteria
-only a small fraction of the bacteria will have taken up the plasmid containing the desired gene
-these can be identified via markers
-those that have not taken up the desired gene are destroyed
-remaining bacteria are cultured
-every time a bacterium divides the desired gene is cloned
Recombinant DNA can be used to produce recombinant proteins (RP)
Bacteria have been genetically engineered for the production of human protein insulin to treat diabetes
Uses of Recombinant DNA Technology
DNA that has been altered by introducing nucleotides from another source is called recombinant DNA (rDNA)
If the organism contains nucleotides from a different species it is called a transgenic organism
Any organism that has introduced genetic material is a genetically modified organism (GMO)
Recombinant DNA has been used to produce recombinant proteins (RP), thus recombinant proteins are manipulated forms of the original protein
Producing recombinant proteins
Recombinant proteins are generated using microorganisms such as bacteria, yeast, or animal cells in culture. They are used for research purposes and for treatments (eg. diabetes, cancer, infectious diseases, haemophilia)
Most recombinant human proteins are produced using eukaryotic cells (eg. yeast, or animal cells in culture) rather than using prokaryotic cells, as these cells will carry out the post-translational modification (due to the presence of Golgi Apparatus and/or enzymes) that is required to produce a suitable human protein
The advantages of genetic engineering organisms to produce recombinant human proteins are:
More cost-effective to produce large volumes (i.e. there is an unlimited availability)
Simpler (with regards to using prokaryotic cells)
Faster to produce many proteins
Reliable supply available
The proteins are engineered to be identical to human proteins or have modifications that are beneficial
It can solve the issue for people who have moral or ethical or religious concerns against using cow or pork produced proteins
Insulin
In 1982, insulin was the first recombinant human protein to be approved for use in diabetes treatment
Bacteria plasmids are modified to include the human insulin gene
Restriction endonucleases are used to cut open plasmids and DNA ligase is used to splice the plasmid and human DNA together
These recombinant plasmids are then inserted into Escherichia coli by transformation (bath of calcium ions and then heat or electric shock)
Once the transgenic bacteria are identified (by the markers), they are isolated, purified and placed into fermenters that provide optimal conditions
The transgenic bacteria multiply by binary fission, and express the human protein - insulin, which is eventually extracted and purified
The advantages for scientists to use recombinant insulin are:
It is identical to human insulin, unless modified to have different properties (eg. act faster, which is useful for taking immediately after a meal or to act more slowly)
There is a reliable supply available to meet demand (no need to depend on availability of meat stock)
Fewer ethical, moral or religious concerns (proteins are not extracted from cows or pigs)
Fewer rejection problems or side effects or allergic reactions
Cheaper to produce in large volumes
That it is useful for people who have animal insulin tolerance
Factor VIII
Factor VIII is a blood-clotting protein that haemophiliacs cannot produce
Kidney and ovary hamster cells have been genetically modified to produce Factor VIII
Once modified these recombinant cells are placed into a fermenter and cultured
Due to the optimal conditions in the fermenter, the hamster cells constantly express Factor VIII which can then be extracted and purified, and used as an injectable treatment for haemophilia
The advantages for scientists to use recombinant Factor VIII are:
Fewer ethical, moral or religious concerns (proteins are not extracted from human blood)
Less risk of transmitting infection (eg. HIV) or disease
Greater production rate
Gene therapy
The use of gene therapy in medicine is becoming more common
Gene therapy involves using various mechanisms to alter a person’s genetic material to treat, or cure, diseases
As scientists gain a better understanding of the human genome and therefore the location of genes that cause genetic disorders, the possibilities of gene therapy being able to replace a faulty gene, inactivate a faulty gene or insert a new gene are growing
Most gene therapies are still in the clinical trial stage because scientists are having difficulty finding delivery systems that can transfer normal alleles into a person’s cells and how to ensure the gene is correctly expressed once there
Gene therapy is being used in medicine for introducing corrected copies of genes into patients with genetic diseases (eg. cystic fibrosis, haemophilia, severe combined immunodeficiency)
Currently, all gene therapies have targeted and been tested on somatic (body)
Changes in genetic material are targeted to specific cells and so will not be inherited by future generations (as somatic gene therapy does not target the gametes)
Often the effects of changing the somatic cells are short-lived
There are two types of somatic gene therapy:
Ex vivo – the new gene is inserted via a virus vector into the cell outside the body. Blood or bone marrow cells are extracted and exposed to the virus which inserts the gene into these cells. These cells are then grown in the laboratory and returned to the person by injection into a vein
In vivo – the new gene is inserted via a vector into cells inside the body
There is the potential for new genetic material to be inserted into germ cells (cells involved in sexual reproduction eg. gametes or an early embryo)
However, this is illegal in humans as any changes made to the genetic material of these cells is potentially permanent and could therefore be inherited by future generations
Severe combined immunodeficiency (SCID)
Severe combined immunodeficiency (SCID) is caused by the body’s inability to produce adenosine deaminase (ADA), an enzyme that is key to the functioning of the immune system. Without this enzyme, children can die from common infections and therefore need to be kept isolated often inside plastic ‘bubbles’
To treat SCID scientists have used ex vivo somatic gene therapy. During this therapy, a virus transfers a normal allele for ADA into T-lymphocytes removed from the patient and the cells are then returned via an injection
This is not a permanent cure as the T-lymphocytes are replaced by the body over time and therefore the patient requires regular transfusions every three to five months to keep their immune systems functioning
Originally retroviruses were used as the vectors, however these viruses insert their genes randomly into a host’s genome which means they could insert the gene into another gene or into a regulatory sequence of a gene (which could result in cancer)
Initial treatments did cause cases of leukaemia in children, so researchers switched to using lentiviruses or adeno-associated viruses as vectors. Lentiviruses also randomly insert their genes into the host genome however they can be modified to not replicate, whereas adeno-associated viruses do not insert their genes into the host genome and therefore the genes are not passed onto the daughter cells when a cell divides. This is an issue with short-lived cells like lymphocytes but has not been a problem when used with longer living cells such as liver cells
Social & ethical considerations associated with gene therapy
The potential for side effects that could cause death (eg. the children who were treated for SCID developed leukaemia)
Whether germline gene therapy (the alteration of genes in egg and sperm cells which results in the alteration being passed onto future generations) should be allowed – it could be a cure for a disease or it could create long-term side effects
The commercial viability for pharmaceutical companies – if it is a rare disease, the relative small number of patients may not mean that the companies will make a profit (eg. Glybera – a gene therapy for lipoprotein lipase deficiency is no longer produced as there were too few patients)
The expense of treatments as multiple injections of the genes may be required if the somatic cells are short-lived (eg. severe combined immunodeficiency). This may make the cost of gene therapy accessible to a limited number of people
The possibility that people will become less accepting of disabilities as they become less common
Who has the right to determine which genes can be altered and which cannot (eg. should people be allowed to enhance intelligence or height)
Another method of enhancing sporting performances unfairly through gene doping. This is where the genes are altered to give an unfair advantage eg. to provide a source of erythropoietin (the hormone that promotes the formation of red blood cells)
Genetic engineering in agriculture
Although plants and animals have been genetically engineered to produce proteins used in medicine, the main purpose for genetically engineering them is to meet the global demand for food
Crop plants have been genetically modified to be:
Resistant to herbicides – increases productivity / yield
Resistant to pests – increases productivity / yield
Enriched in vitamins – increases the nutritional value
Farmed animals have been genetically modified to grow faster. It is rarer for animals to be modified for food production due to ethical concerns associated with this practice
Scientists have genetically modified many organisms including bacteria (eg. to produce insulin), sheep (eg. to produce a human blood protein known as AAT), maize (eg. to be resistant to insect attacks), rice (eg. to produce β-carotene to provide vitamin A)
The benefits of using genetic engineering rather than the more traditional selective breeding techniques to solve the global demand for food are:
Organisms with the desired characteristics are produced more quickly
All organisms will contain the desired characteristic (there is no chance that recessive allele may arise in the population)
The desired characteristic may come from a different species / kingdom
GM salmon
-salmon that has been genetically modified (GM) to grow more rapidly than non-GM salmon
-due to growth hormone being produced in the salmon throughout the year, instead of just in spring and summer
-producer gets higher yield to can make more profit
-scientists combined a growth hormone gene from a chinook salmon with the promoter gene from an ocean pout, a cold-water fish
-ocean pout can grow in near-freezing waters
-hence the promoter gene ensured the growth hormone was continually being expressed
-all GM salmon are female and sterile
-prevent reproduction in the wild
Insect resistance in cotton
-cotton has been genetically modified with a gene for the Bt toxin (taken from the bacterium Bacillus thuringiensis)
-Bt toxin gene allows cotton plants to produce their own insecticide
-when an insect ingests parts of the cotton plant, the alkaline conditions in their guts activate the toxin
-kills insect
-the toxin is harmless to vertebrates as their stomach is highly acidic
-different strains of thuringiensis produce different toxins which are toxic to different insect species
-insect populations have developed resistance to the genes for Bt toxin
-reduces its effectiveness as a means of protecting crops
Social and ethical implications associated with GMOs in agriculture
The genetic modification of microorganisms for the production of medicines, antibiotics and enzymes raises little debate compared to the use of genetically modified organisms (GMOs) for food production
The use of GMOs in food production has been proposed as a solution to feeding the increasing world population, the decreasing arable land and decreasing the impact on the environment, however concerns such as the development of resistance in insects and weeds and costs of seeds have meant that countries are not allowing GMOs to be grown
The solution could be integrated pest-management systems that could help avoid the development of resistance and increased population of secondary pests
The ethical implications of using GMOs in food production are:
The lack of long-term research on the effects on human health – should GM food be consumed if it is unknown whether it will cause allergies or be toxic over time (although there has been no evidence to suggest this would occur to date)
Making choices for others:
That without appropriate labelling the consumer cannot make an informed decision about the consumption of GM foods
As the pollen from the GM crop may contaminate nearby non-GM crops that have been certified as organic
By reducing the biodiversity for future generations
social implications of growing GMOs for food
The social implications of growing GMOs for food evolve around whether the crops are safe for human consumption and for the surrounding environment
The possible implications are:
The GM crops may become weeds or invade the natural habitats bordering the farmland
The development of resistance for the introduced genes in the wild relative populations
Potential ecological effects (e.g. harm to non-targeted species like the Monarch butterflies)
Cost to farmers (new seed needs to purchase each year)
The ability to provide enriched foods to those suffering from deficiencies (eg. Golden Rice) and therefore decrease diseases
Reduced impact on the environment due to there being less need to spray pesticides (eg. less beneficial insects being harmed)
Reduction in biodiversity which could affect food webs
The herbicides that are used on the GM crops could leave toxic residues
Define DNA hybridisation
DNA probes
-process that is used in medical diagnostic tests and genetic screening
-two complementary single-stranded DNA molecules combine through base pairing
-forms a single double-stranded DNA molecule
DNA probe:
-short length of single-stranded DNA
-has a known base sequence complementary to the specific base sequence of a known allele
-DNA probes are used in conjunction with DNA hybridisation
-can indicate whether specific harmful alleles are present in a DNA sample
-probe is usually attached to a radioactive or fluorescent label that indicates its position
-part of the base sequence of the harmful allele must be known
-enables DNA probe to be synthesised using a gene machine
Benefit of genetic screening?
encourage individuals to make lifestyle choices to help prevent disease or provide them with information for viable treatment options
Using DNA probes to locate specific alleles of genes
-cell sample is taken from a patient
-DNA is extracted from the cell sample and purifified
-PCR is used to amplify test DNA (as cell sample produces a small amount of purified DNA)
-PCR produces identical copies of the DNA
-restriction endonucleases are used to digest the amplified test DNA
-done because whole DNA molecules are too long to be analysed in a single go
-result: restriction fragments that are separated using gel electrophoresis
-negatively charged DNA fragments move through the pores in the gel, towards the positive electrode
-smaller DNA fragments are able to move at a faster rate through the pores
-hence they travel a further distance
-fragments separate according to size and charge, producing bands in the gel
-bands of DNA are transferred to a nylon membrane
-hydrogen bonds between complementary base pairs are broken
-makes DNA fragments on the nylon membrane single-stranded
-labelled DNA probes are added to the nylon membrane
-these DNA probes have a specific base sequence
-complementary to that of the harmful allele (it must not be complementary to any normal alleles)
-DNA on the nylon membrane is single-stranded
-hence probes can anneal to any complementary DNA fragments present
-nylon membrane is washed to remove any excess DNA probes
-processed to reveal the position of any bound DNA probes
-for fluorescent labels, UV light is used to detect their position
-radioactive labels: autoradiography is used to detect their position
-if the label shows up on any of the restriction fragments present on the nylon membrane, then the DNA in that particular position must be from the harmful allele
-if no labels show up then the test DNA does not contain the harmful allele of the gene
Limitation of DNA probes
-often only tests for one specific harmful allele
-individual may produce a negative test result for that specific harmful allele
-but they could have another more rare harmful allele, caused by different mutations in their DNA
Genetic Screening
Certain circumstances may require individuals to determine if they have a particular allele present in their genome
-e.g. in the pregnancy in an older woman
-pregnancy where there is a family history of a genetic disease)
-can be determined by genetic screening
-identifies individuals who are carrying an allele at a gene locus for a particular disorder
-sample of DNA to be analysed can be obtained by:
-taking tissue samples from adults or embryos produced by in-vitro fertilisation
-Chorionic villus sampling or amniocentesis of embryos and fetuses in the uterus
Breast cancer (BRCA1 and BRCA2)
BRCA1 and BRCA2 are genes that produce tumour suppressor proteins and thus they play an important role in regulating cell growth
Faulty alleles of these particular genes exist which increase the risk of an individual developing breast and ovarian cancers during their lifetime
Faulty BRCA1 and BRCA2 alleles can be inherited from either parent
The advantages of genetic screening for an adult who has a family history of BRCA1 and BRCA2 gene mutations are:
That the person may decide to take preventative measures (e.g. by having an elective mastectomy – breast removal – to reduce the risk of developing cancer)
Screening for breast cancer may begin from an earlier age or more frequently, and the individual (if female) will have more frequent clinical examinations of the ovaries
That it enables the person to participate in research and clinical trials
Huntington’s disease
Huntington’s is a progressive (gets worse with time) inherited disease that affects the brain
Signs of the disease typically appear in affected individuals after reaching their 40’s and include uncontrolled movements, lower cognitive (thinking) ability and emotional problems
There is no cure for the Huntington’s disease, with treatments available only alleviating the symptoms but not curing it
Huntington’s is an autosomal dominant disease (therefore if the person has an allele for Huntington’s they will get the disease)
The advantage of genetic screening for Huntington’s is it enables:
People to plan for the future (how they will live and be cared for)
Couples to make informed reproductive decisions (as the risk that their children may inherit the disease is 50%)
People to participate in research and clinical trials
Cystic fibrosis
Cystic fibrosis is an autosomal recessive genetic disorder that is caused by a mutation of the gene that codes for a transported protein called CFTR
It is a progressive disease that causes mucus in various organs (lungs, pancreas, lungs) to become thick and sticky. This is because the faulty CFTR protein no longer transports chloride ions across the cell plasma membrane and therefore water does not move by osmosis across the membrane either (the presence of water would normally make the mucus thinner enabling cilia to remove it)
There is no cure for cystic fibrosis, although there are many different treatments that help alleviate symptoms. The common cause of death is bacterial infection in the lungs
The advantage of genetic screening for cystic fibrosis is:
It enables couples to make informed reproductive decisions (as both may be carriers and therefore not display any symptoms)
That people can participate in research and clinical trials
Evaluating the Use of Genetic Screening
Arguments For Genetic Screening
Screening for certain conditions can enable people to make sensible lifestyle choices to reduce the chances of the disease developing
For example, if you are found to be genetically predisposed to getting cancer or developing heart disease, you can decide to eat a healthy diet and to refrain from smoking in order to reduce the chances of these diseases developing
Screening enables potential parents to choose whether or not to have their own biological children, as they may not want to risk passing on a harmful allele
Screening enables people to participate in research and clinical trials, which are critical for developing the understanding of and treatments for genetic disorders
Arguments Against Genetic Screening
Screening people for an incurable disease or one that develops in later life may not be beneficial as there may be nothing positive that can be done in response to this information, potentially leading to the person becoming depressed or scared
This may also lead to someone having to pay a higher price for life insurance (which they may not be able to afford) than other people
Some people fear that screening may lead to a form of genetic discrimination against individuals with defective or disease-causing alleles (which may be seen by some people as being “inferior”, although this is not the case and no-one should ever be seen as being genetically inferior)
In addition, parents found to have a high chance of passing on harmful alleles may be unfairly pressured into not having children
Some people fear that screening may one day be used to look more broadly at the genetic make-up of a potential child (i.e. not just focusing on the risks of genetic disorders developing), which could eventually lead to potential parents making reproductive decisions such as aborting foetuses that do not have the desired genetics
This idea of ‘designer babies’ becoming a possibility in the future raises many ethical questions
Some religions consider such interferences with the natural process of reproduction to be highly immoral
Genetic counselling
-commonly used to help individuals understand and process their results
-counsellors can answer their questions
-can also be seen before a screening has occurred to inform an individual of the possible results
They may discuss the following with a patient:
-chances of the individual developing an inherited disease
-lifestyle changes that can be made to reduce or manage the risk of developing the disease
-therapeutic treatments
-chance of an individual having a child with a certain disease
-termination of a pregnancy
-financial implications of having a child with severe disabilities
-ethical issues
Personalised medicine
-involves development of more targeted drugs
-treats a variety of human diseases and the development of synthetic tissues
-genomic medicine can be developed using information gathered from genome projects
-e.g. Human Genome Project (HGP)
-genomic medicine uses information about an individuals genes to influence their clinical care
-genetic screening = individuals with a high chance of developing specific diseases are identified
-preventative measures or precautions to be taken
variable number tandem repeats
Variable number tandem repeats (VNTRs) are regions found in the non-coding part of DNA
A short DNA sequence (e.g. GATA) is repeated a variable number of times without any spaces, at a single location
These VNTRs may be referred to as ‘satellite’ or ‘microsatellite’ DNA
The length of VNTRs varies between different people
The probability that two individuals would have the same length VNTRs is extremely small
As a result, VNTRs can be used to identify the source of DNA from tissue samples
However, the number of VNTRs a person has is inherited
They can be used to identify biological parents
Different restriction enzymes are used to cut the DNA at different base sequences close to the variable number tandem repeat (VNTR) regions
The distance between recognitions sites of a restriction enzyme will change depending on the number of repeated sequences
Therefore DNA fragments of different lengths are produced when the same restriction enzymes are applied to different DNA samples
When these DNA fragments undergo electrophoresis they produce a distinctive pattern
Only identical twins will produce an identical pattern of restriction fragments
variable number tandem repeats 2
apparatus for gel electrophloresis
Agarose gel
Electrophoresis tank
Electrolyte solution
Micropipette
Electrodes
DNA sample
DNA standard
Probes
Nitrocellulose
A dye
method for gel electrophloresis
Create an agarose gel plate in a tank. Cut wells into the gel at one end
-different gels have different sized pores
-affects speed of molecules when moving through them
Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
Pipette the DNA samples into the wells using a micropipette ensuring the DNA standard is loaded into the first well
After collection, DNA fragments are prepared
-scientists must purify the DNA, increase (amplify) the number of DNA molecules by the polymerase chain reaction (PCR)
-restriction endonucleases (enzymes) are used to cut the DNA into fragments
Connect the negative electrode to the end of the plate with the wells and connect the positive anode at the far end
The DNA fragments will then move towards the anode (positive pole) due to the attraction between the negatively charged phosphates of DNA and the anode
The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which is then heated to separate the two DNA strands
Probes are then added, after which an X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is generally compared to a control fragment of DNA
Probes are single-stranded DNA sequences that are complementary to the VNTR regions sought by scientists. The probes also contain a means by which to be identified. This can either be:
A radioactive label (eg. a phosphorus isotope) which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands
A fluorescent dye (eg. ethidium bromide) which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands
limitations for gel electrophloresis
-measurements are not precise
-must be compared to a standard in order to gather data
-electrophoresis requires a lot of sample
-depends on PCR
-amplifies DNA fragments
why is gel electrophloresis used
technique used widely in the analysis of DNA, RNA and proteins
During electrophoresis the molecules are separated according to their size / mass and their net (overall) charge
Of the electrical charge molecules carry – positively charged molecules will move towards the cathode (negative pole) whereas negatively charged molecules will move towards the anode (positive pole) eg. DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move towards the anode
Different sized molecules move through the gel (agarose for DNA and polyacrylamide – PAG for proteins) at different rates. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly
Different restriction enzymes cut the DNA at different base sequences. Therefore scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions
Variable number tandem repeats (VNTRs) are regions found in the non-coding part of DNA. They contain variable numbers of repeated DNA sequences and are known to vary between different people (except for identical twins). These VNTR may be referred to as ‘satellite’ or ‘microsatellite’ DNA
Genetic Fingerprinting
DNA can be collected from almost anywhere on the body, e.g. the root of a hair or saliva from a cup. After collection DNA must be prepared for gel electrophoresis so that the DNA can be sequenced or analysed for genetic profiling (fingerprinting)
Fingerprinting can be used to determine genetic relationships and the genetic variability within a population
To prepare the fragments scientists must first increase (amplify) the number of DNA molecules by the polymerase chain reaction (PCR). Then restriction endonucleases (enzymes) are used to cut the DNA into fragments
Different restriction enzymes cut the DNA at different base sequences. Therefore scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions
Variable number tandem repeats (VNTRs) are regions found in the non-coding part of DNA. They contain variable numbers of repeated DNA sequences and are known to vary between different people (except for identical twins). These VNTRs may be referred to as ‘satellite’ or ‘microsatellite’ DNA
DNA separation by gel electrophoresis
Gel electrophoresis is a technique used widely in the analysis of DNA, RNA and proteins. During electrophoresis the molecules are separated according to their size / mass and their net (overall) charge
The separation occurs because:
Of the electrical charge molecules carry – positively charged molecules will move towards the cathode (negative pole) whereas negatively charged molecules will move towards the anode (positive pole) eg. DNA is negatively charged due to the phosphate groups and thus when placed in an electric field the molecules move towards the anode
Different sized molecules move through the gel (agarose for DNA and polyacrylamide – PAG for proteins) at different rates. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly
Of the type of gel – different gels have different sized pores which affects the speed the molecules can move through them
Variable number tandem repeats
-DNA profiling (genetic fingerprinting) enables scientists to identify suspects for a crime and identify corpses
-every person (apart from identical twins) has repeating short non-coding regions of DNA that are unique to them
-20 to 50 bases
-called variable number tandem repeats (VNTRs)
-number of VNTR regions are inherited from your biological parents
-more closely related you are to a person the more likely the repeats have similar patterns
-length of the VNTR regions are unique to each individual (apart from identical twins)
-when DNA testing occurs in forensic medicine and criminal investigations the image of these repeats in the DNA (indicated by a pattern of bars) creates a DNA profile or fingerprint
-profile is analysed to draw conclusions
DNA profile is created with this method:
1) Obtain the DNA from the root of a hair, a spot of blood or semen or saliva
2) Increase the quantity of DNA by using PCR
3) Use restriction endonucleases to cut the amplified DNA molecules into fragments
-different restriction endonucleases cut close to different VNTR sequences
4) Separate the fragments using gel electrophoresis
5) Add radioactive/fluorescent probes that are complementary, hence bind to specific VNTR regions
6) X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing
-images contain patterns of bars (the DNA profile) which are then analysed
Uses of genetic fingerprinting
Forensic medicine / criminal investigations
-DNA profiling has been used to identify suspects of crimes
-samples of body cells or fluids (eg. blood, salvia, hair, semen) are taken from the crime scene or victims body
-DNA is removed and profiled
-profile is compared to samples from:
-the suspect (or criminal DNA database)
-victim
-people with no connection to the crime (control samples)
-care must be taken to avoid contamination of the samples
-DNA profiling can also be used in forensics to identify bodies or body parts that are unidentifiable
Other applications
-identifying individuals that are at risk of developing particular diseases
-research shows that certain VNTR sequences are associated with an increased incidence of particular diseases
-e.g. cancers and heart disease
-determining familial relationships for paternity cases or immigration cases
Species conservation
-helps scientists with captive breeding programmes
-reduces chances of inbreeding
What are carcinogens
-agents that may cause cancer
-UV light
-tar in tobacco smoke
-X-rays
in vivo transformations
-restriction endonucleases are used to cut DNA into fragments with sticky ends
-a promoter and termination region are added (allows transcription)
-plasmid is cut open with same restriction endonuclease
-creates complementary sticky ends
-DNA loop in bacteria acts as a vector
-DNA ligase is used to incorporate DNA fragments into plasmid
-recombinant DNA is formed
-recombinant plasmid is transferred to bacteria
finish fc
