Manipulating genomes Flashcards
Cloning DNA
-gene to be sequenced isolated using restriction enzymes
-DNA inserted into bacterial plasmid (vector)
-plasmid inserted into E.coli bacteria
-E.coli cultured- divided many times
-each new bacteria contained copy of candidate/ desired gene
-length of DNA isolated using plasmid preparation techniques
=also known as a clone library
Describe DNA sequencing process
SEQUENCING MIXTURE
-many copies of original DNA, need to be single stranded
-primers
-DNA polymerase
-DNA free nucleotides- some of which may have fluorescent markers which throw off DNA polymerase and prevent more free nucleotides being added
PROCESS
1) many copies of single stranded template DNA fragment
2)primers added- anneal to 3’ end enabling DNA polymerase to attach
3) DNA polymerase extends primer with DNA free nucleotides (ordinary) until modified/ fluorescent marked DNA free nucleotide added - this throws off DNA polymerase and stops extension
4) terminator base is marked by fluorescent tag
5) results in many different lengths of DNA, each tagged at end by fluorescent marker
6) transferred to electrophoresis machine- shortest base fragments arrive first, their tag is read by laser
7) computer records sequence of bases
Bioinformatics
-branch of biology that deals with storing, displaying and using large quantities of often complex biological data
-combines computer science, statistics, maths and biology
-aids molecular, structural and function analysis of genes, genomes and their products
-used in medicine, personalised drugs, gene therapy, insect resistance, evolutionary studies, alternative energies etc
-bioinformatics decreases time taken for development
What was discovered as a result of human genome project
-HGP launched 1990, genome was sequenced by 2003
-scientists learnt human genome contained only about 24,000 genes
-work continues trying to sequence other species
-much collaboration, sharing information, peer reviewing data to check it, open access
Genome comparisons between individuals and species
-whole genome sequencing determines complete DNA sequence of an organisms genome- in case of eukaryotic cells that is the genetic material of chromosomes, mitochondria and chloroplasts
-sequenced genomes are stored in gene banks
Comparison between species
-when human genome was compared with those of other species it became clear that few human genes are unique to us
-most of our genes have counterparts in other organisms
-we share 99% of our genes with chimpanzees
-this verifies that genes work well tend to be conserved by evolution
-sometimes as evolution progresses some genes are coopted to perform new tasks
-tiny changes to gene in humans called FOXP2 allows speech
-many of the differences between organisms are not because the organisms have totally different genes but because some of their shared genes have been altered and now work in subtly different ways
-some changes to regulatory regions of DNA that do not code directly for proteins have also altered expression of genomes- regulatory and coding genes interact in such ways that without increasing number of genes, the number of proteins may be increased
-we can look for evolutionary relationships through interspecific relationships and build phylogenetic trees
Variation between individuals
-all humans are genetically similar
-except for rare cases where a gene has been lost by depletion of part of a chromosome we all have the same genes, but different alleles
-about 0.1% of our DNA is not shared with others
-the places on DNA where these substitutions occur are called single nucleotide polymorphisms or SNPs
-some have no effect on the protein, some can alter a protein or alter the way a piece of RNA regulates the expressions of another gene
-methylation of certain chemical groups in DNA plays a major role in regulating gene expression in eukaryotic cells
-methods to map to methylation of whole human genomes can help researchers understand development of certain diseases, for example cancer and why they may or may not develop in genetically similar individuals
-the study of this aspect of genetics is called epigenetics
-useful to compare genomes of individuals: find out which genes susceptible to, which medicines give side effects (personalised medicine), genetic profiling (e.g. at crime scene, paternity tests)
Predicting amino acid sequences of proteins
-determining sequence of amino acids within protein is laborious and time consuming
-however if researchers have organisms genome sequenced and know which gene codes for a specific protein by using knowledge of which base triplets code for which amino acids (aka genetic dictionary), they can determine the primary structure of proteins
-researchers need to know which part of gene codes for exons and which codes for introns
-can predict shape of proteins and use them to make complementary drugs (personalised medicine)
Synthetic biology
-synthetic biology is an interdisciplinary science concerned with designing and building useful biological devices and systems
-its ultimate goal may to build engineered biological systems that store and process information, provide food, maintain human health and enhance environment
-BIOTECHNOLOGY= genetic engineering of an organisms e.g. transgenic goats, golden rice, BT cotton, antibiotics, medicine production, gene therapies biosensors
-EVOLUTIONARY BIOLOGY= look at interspecific comparisons to form phylogenetic trees
-MOLECULAR BIOLOGY= look at interaction between DNA, RNA and proteins. Sequences DNA analysed for making synthetic biology devices e.g. synthetic DNA to store information
-SYSTEMS BIOLOGY= mathematical and computer modelling of complex biological systems e.g. cell signalling, bionomics, epigenetics
-BIOPHYSICS= uses physic techniques to look at biological systems e.g. biofilms are amyloid fibres that can be used for functions such as adhesions, however threat of infection
-BIOETHICS= raises issues of biosecurity
Examples of synthetic biology
-information storage= scientists can encode a vast amount digital information onto single strand of synthetic DNA
-production medicines= E.coli and yeast have both been genetically engineered to produce precursor of good antimalarial drug, only available by extracting it from certain parts of Artemisia plants at particular times to its life cycle
-novel protein= designed proteins produced for example one similar to haemoglobin and binds to oxygen but not to carbon monoxide
-biosensors= modified bioluminescent bacteria, placed on a coating of microchip, grow if air polluted with petroleum pollutants
-nanotechnology= material can be produced for nanotechnology e.g. amyloid fibres for making biofilms - for functions such as adhesion
Bioethics
-synthetic biology raises issues of ethics and biosecurity
-extensive regulations are already in place, due to 30-40 years of using genetically modified organisms
-there are many advisory panels and many scientific papers have been written on how to manage risks
-synthetic biology is not about making synthetic life forms from scratch but about potential for new systems with rewards and associated risks to be managed
Development of DNA profiling
-Alec Jeffreys was locating tandem repeat sequences of DNA
-tandem repeats are repetitive sequences of DNA that do not code for proteins
-they may be between 10 and 100 base pairs long and they all feature the same core sequences GGCAGGAXG where X can be any of the four nucleotide bases
-tandem repeats occur at more than 1000 locations in genome and in each of these places they may be repeated a random number of times
-some types are highly variable and called variable number tandem repeats (VNTRs)
DNA profiling procedure
1) Data is obtained from the individual- either by mouth swab, from saliva on tooth brush, blood (WBC as RBS have no nucleus therefore no DNA) or hair or in case of ancient remains, bone
2) DNA is then digested with restriction enzymes. These enzymes cut the DNA at specific recognition sites. They will cut into fragments which will vary in size to each individual
3) the fragments are separated by gel electrophoresis and stained. larger fragments travel shortest distance in gel
4) a banding pattern can be seen
5) DNA to which individuals is being compared is treated with same restriction enzymes and subjected to electrophoresis
6) the banding patterns of DNA samples can then be compared
Types of DNA analysed
-the first method involved in restriction fragment length polymorphism analysis- method is no longer used as is laborious
-today short tandem repeat (STR) sequences of DNA are used
-these are highly variable short repeating length of DNA
-the exact number of STR varies from person to person
-each STR is polymorphic but the number of alleles in the gene pool is small for each one
-technique is very sensitive and even trace of DNA left when someone touches object can produce a result
-samples must be treated carefully to avoid contamination
-DNA can be stored for many years if criminal case unsolved- can later be used to assess new evidence
APPLICATIONS OF DNA PROFILING- forensic science
-DNA profiling has brought about convictions, establish innocence of many people and of people previously wrongly convicted
-e.g. identify nazi war criminals hiding in south america
-identify victims body parts after air crashes, terrorist attacks etc
-identify individuals remains
APPLICATIONS OF DNA PROFILING- maternity and paternity tests
-half of every child genetic information comes from mother and half from father
-hence short tandem repeat fragments come from mother and father
-comparing DNA profiles of mother, father and child can therefore establish maternity and/or paternity
APPLICATIONS OF DNA PROFILING - analysis of disease
-protein electrophoresis can detect type of haemoglobin present and aid diagnosis of sickle cell anaemia
-a varying number of repeat sequences for condition such as Huntington disease can be detected by electrophoresis
Principles of the polymerase chain reaction (PCR)
-Kay Mullis developed the technique the polymerase chain reaction to amplify (increase amount) of DNA, enabling analysis
-PCR soon became incorporated into forensic DNA analysis and into protocols for analysis of DNA for genetic diseases
-the PCR is artificial replication of DNA- it relies upon:
-DNA is made of two antiparallel backbone strands
-each DNA strand has a 5’ end and 3’ end
-DNA grows only from the 3’ end
-base pairs pair up according to complementary base pairing rules A with T, G with C
How does PCR differ from DNA replication
-only short sequences of up to 10,000 base pairs of DNA can be replicated not entire chromosomes
-it requires addition of prime molecules to make process start
-cycle of heating and cooling is needed to separate DNA strands, bind primers to strands and for DNA strands to be replicated
PCR process
1)heat reaction mixture to 95C
-this splits the double stranded original DNA into a single strand
-the hydrogen bonds are broken
2)temperature is lowered to 68C
-primers anneal to 3’ end of each single strand
-DNA polymerase cannot be used as it cannot bind to the single strands
-hydrogen bonds form
3) temperature raised to 72C
-this is optimum temperature for Taq DNA polymerase enzyme
-Taq DNA polymerase binds to double stranded section (3’ end) and extends primer by adding DNA free nucleotides- catalyses formation phosphodiester bonds
4) results in formation of two copies of original DNA
-whole process beings again and repeats for many cycles; amount DNA increases exponentially 1-2-4-8-16-32-64-128 etc
Applications of the PCR
-tissue typing: donor and recipient tissues can be typed prior to transplantation to reduce risk of rejection of transplant
-detection of oncogenes: if type mutation involved in specific patient cancer found, medication better tailored to that patient
-detecting mutations: sample DNA analysed for presence of mutation that leads to genetic disease. Parents can test to see if carry recessive allele for particular gene e.g. fetal cells may be obtained from mothers blood stream for prenatal genetic screening
-identifying viral infections: sensitive PCR tests can detect small quantity of viral genome amongst host cell DNA e.g. for HIV, hepatitis C
-monitoring spread infectious disease: spread of pathogens through wild or domestic populations or from animals to humans can be monitored and emergence of new virulent sub types can be identified
-forensic science: small quantities of DNA can be amplified for DNA profiling to identify criminals or ascertain parentage
-research: amplifying DNA from extinct ancient sources for analysis and sequences
ELECTROPHORESIS: separating DNA
-electrophoresis is used to separate different sized fragments of DNA
-it can separate fragments that differ only by one base pair and is widely used in gene technology to separate DNA fragments for analysis and identification
-the technique uses and agarose gel plate covered by buffer solution
-electrodes are placed in each end of tank so when connected to power supply and electric current can pass through the gel
-DNA has an overall negative charge due to its many phosphate groups and the fragments of DNA all have similar surface charge regardless of size
ELECTROPHORESIS: separating proteins
-principle for separating proteins is same as for separating fragments of DNA but often carried out in presence of charged detergent such as sodium dodecyl sulfate (SDS) which equalises surface charge on molecules and allows proteins to separate as they move through the gel according to their molecular mass
What can separating proteins be used for
-technique can be used to analyse types of haemoglobin proteins for diagnosis of conditions such as
-sickle cell anaemia where patient has haemoglobin S not normal A
-aplastic anaemia, thalassaemia and leukaemia where patients have higher than normal amounts of fetal haemoglobin (F) and lower than normal amounts of haemoglobin A
Using DNA probes
-a DNA probe is a short single stranded length of DNA that is complementary to section of DNA being investigated (50-80 nucleotides)
-probe may be labelled using
-a radioactive marker usually with 32P in one of phosphate groups in probe strand
-once probe has annealed by complementary base pairing to piece of DNA it can be revealed by exposure to photographic film
-or a fluorescent marker that emits a colour on exposure to UV light
-fluorescent markers may also be used in automated DNA sequencing
What are DNA probes used for
-useful in locating specific DNA sequences e.g.
-to locate specific gene needed for use in genetic engineering
-to identify same gene in variety of different genomes from different species when conducting genome comparison studies
-to identify presence or absence of specific allele for particular genetic disease or that fives susceptibility to particular condition
Microarrays
-reference DNA= complementary to alleles known to be present, show you test has worked
-test DNA= complementary to faulty alleles, used in disease diagnosis, locate genes and for genetic engineering
-sample DNA must first be broken into smaller fragments and may also be amplified using PCR
-references and test DNA samples are labelled with fluorescent markers
Principles of genetic engineering
-genetic engineering is also known as recombinant DNA technology because it involves combining DNA from different organisms
-also called genetic modification
-genes are isolated from one organism and inserted to another organism using suitable vectors
1) obtaining the required gene
2) a copy of gene is placed inside vector
3) vector carries gene into recipient cell
4) recipient expresses novel gene
- required gene is obtained
-mRNA can be obtained from cells where the gene is being expressed
-an enzyme reverse transcriptase, can then catalyse the formation of single strand of complementary DNA (cDNA) using mRNA as template
-the addition of primers and DNA polymerase can make this cDNA into double stranded length of DNA
-if scientists know the nucleotide sequence of gene then the gene can be synthesised using automated polynucleotide synthesiser
-if scientists know the sequence of the gene the can design PCR primers to amplify the gene from genomic DNA
-a DNA probe can be used to locate a gene within genome and gene can be cut out using restriction enzymes
- Placing gene into vector
-plasmids can be obtained from organisms such as bacteria and mixed with restriction enzymes that will cut the plasmid at specific recognition sites
-cut plasmid has exposed unpaired nucleotide bases called sticky ends
-if free nucleotide bases complementary to sticky ends of plasmid are added to ends of gene to be inserted then gene cut and plasmid should anneal
-DNA ligase catalyses annealing
-gene may be sealed into attenuated virus that could carry it into host cell
-however may trigger immune system
-vectors often have to contain regulatory sequences of DNA as well in order to express/ turn on the gene
- Getting vector into recipient cell
-DNA does not easily cross recipient cells plasma membrane
-various methods can be used to aid the process:
-heat shock treatment- if bacteria subjected to alternating periods of cold 0C and heat 42C in presence of calcium chloride, their walls and membranes will become more porous and allow in the recombinant vector. This is because positive calcium ions surround negatively charged parts of both DNA molecules and phospholipids in cell membrane, this reducing repulsion between foreign DNA and host cell membranes
-electroporation- high voltage pulse applied to cell to disrupt membrane
-electrofusion- electrical fields help introduce DNA into cells
-transfection- DNA can be packaged into bacteriophage which can then transfect into host cell
-T1 (recombinant) plasmids are inserted into bacterium Agrobacterium tumefaciens which then infects some plants and naturally inserts its genome into host cell genomes
- Direct method introducing gene into recipient
-if plants not susceptible to A. tumefaciens then direct methods can be used
-small pieces of gold or tungsten coated with DNA and shot into plant cells
-this is called a gene gun
Reverse transcriptase
-retroviruses such as HIV which contain RNA that they inject into host genome have reverse transcriptase enzyme that catalyses the production cDNA using their RNA as template
-this is reverse of transcription
-these enzymes are useful for genetic engineering
Restriction enzymes
-bacteria and archaea have restriction enzymes called restriction endonucleases to protect them from attack by phage viruses
-these enzymes cut up foreign viral DNA by a process called restriction preventing the viruses from making copies of themselves
-the prokaryotic DNA is protected from action of endonucleases by being methylated at recognition sites
-the restriction endonucleases are named according to bacterium from which they have been obtained
-the first used was obtained from E.coli
-restriction enzymes are useful to molecular biology and biotechnology as molecular scissors as they recognise specific sequences within length of DNA and cleave molecular there
-some make a staggered cut leaving sticky ends
-other make a cut that leaves blunt ends
-the enzymes always recognise a palindromic sequence; reading two strands of DNA in same orientation, sequences of bases is same
-some restriction endonucleases need magnesium ions as cofactors
Ligase enzymes
-DNA ligase enzymes used in molecular biology to join DNA fragments
-it catalyses condensation reactions that join sugar groups and phosphate groups of DNA backbone
-these enzymes catalyse such reactions during DNA replication in cells and are also used in PCR
Insulin from GM bacteria
1)adding reverse transcriptase enzymes makes single strand of cDNA and treatment with DNA polymerase makes double strand- the gene
2) addition of free unpaired nucleotide at end of DNA produces sticky ends
3) now with help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bacteria
-these are now called recombinant plasmids as they contain inserted DNA
4)E.coli bacteria are mixed with recombinant plasmids and subjected to heat shock in presence of calcium chloride ions so they will take up plasmids
Safety of genetic engineering
-because transformed/ transgenic bacteria have resistance to some antibiotics we don’t want them to escape laboratories into wild
-they also have gene knocked out meaning they cannot synthesise particular nutrient
-they survive in lab where they are given that nutrient in growth medium but will not survive outside these conditions
ISSUES GENETIC MANIPULATION: microorganisms
+GM microorganisms can make human insulin to treat all diabetics (not possible with pig insulin)
+make human growth hormone to treat children with pituitary dwarfism
-microorganism could escape into wild and transfer marker genes for antibiotic resistance to other bacteria - HOWEVER, cannot synthesise essential nutrient so will not survive outside lab
ISSUES GENETIC MANIPULATION: plants
+Beta carotene gene in rice used to treat vitamin A deficiency and prevent blindness e.g. Golden rice used in India where there is lack of vitamin A
+resistance to pests therefore need to use fewer pesticides- fewer associated risks e.g. neonicotinoids kill bees
+resistance to herbicides; can spray crop without killing it, weeds die and yield greater
+e.g. soya beans resistant to herbicide round up ready
+e.g. bananas enhanced with zinc- treat zinc deficiencies where people eat little meat- is an important enzyme cofactor and essential regulating insulin secretion
-transfer to wild plants resulting in herbicide resistance= superweeds
-toxic to other plants and humans
-transfer pest resistance to wild plants= alter ecosystems
-transfer to wild plants= reduction in genetic variation
-concerns that local farmers have no choice but to buy GM seeds even if they don’t want them
ISSUES GENETIC MANIPULATION: pathogens
+ viruses genetically modified to have no virulence can be used to make vaccines as they still have antigens on their surface- this reduces chance of vaccine making recipient ill
+modified viruses can also be used as vectors in gene therapy
-allele may be used inserted into genome in way that increases risk of cancer or interfere with gene regulation
ISSUES GENETIC MANIPULATION: animals
+GM mice bred for medical research and used to develop therapies for therapies for breast and prostate cancer
+increase yield of milk, creamier milk, milk with less fat, increase egg yield from chickens
+GM pigs express human antigens on surface of cells so when heart valves used for transplant=no rejection
+genes for spider silk inserted into goat- produce spider silk protein in their milk for use in cables, stitches, bullet proof vests etc
+pharmaceutical chemicals produced in milk e.g. alpha anti trypsin in sheep milk treat hereditary emphysema
-moral objections
-animal welfare
-religious views and objections
ISSUES GENETIC MANIPULATION: silk
+gene therapies- e.g. treatment of SCID for ADA deficiency
-offspring not given consent
-germline gene therapy not practised in humans as do not understand potential future side effects
-ethical objections
-designer babies- modern day eugenics
-genetic defects passed to offspring
Principle of gene therapy
-therapeutic technique where functioning allele of certain gene placed in cells of individual lacking said function allele
-if inserted allele is expressed then individual will produce functioning protein and no longer have symptoms associated with genetic disorder
-knowledge gained from Human Genome project has led to further possibilities such as using interference RNA to silence genes by blocking translation
-interference RNA has been used to treat cytomegalovirus in AIDs patients by blocking replication of said virus
Somatic cell gene therapy
-somatic cells are cells with full set of chromosomes, they will not be passed on to offspring
-some metabolic disorders such as cystic fibrosis occur when individual inherits two faulty recessive alleles for particular gene
-as result, differentiated cells where this gene should normally be expressed lack the protein product of that gene
-if functioning alleles for this gene can be put into specific cells so that these cells can make the protein, these cells will function normally
-various methods are available for delivering functioning alleles to the patients body cell
-somatic cell gene therapy affects only certain cell types
-alterations made to patients genome in those cells are not passed to patients offspring
Liposomes
-patients with cystic fibrosis lack functioning CFTR gene
-alleles which are lengths of DNA can be packaged within small spheres of lipid bilayer to make liposomes
-if these are placed into aerosol inhaler and sprayed into noses of patients, some will pass through plasma membrane of cells lining respiratory tract
-if they also pass through nuclear envelope and insert into host genome, host cell will expresses CFTR protein- a transmembrane chloride ion channel
-epithelial cells lining respiratory tract are replaced every 10-14 days so this treatment has to repeated at regular short intervals
-cheaper than viruses, no immune response, especially good for in lung delivery
Viruses
-viruses have been used as vectors
-if virus that usually infects humans is genetically modified so that is encases functioning allele to be inserted into patient whilst at same time being made unable to cause disease, it can enter recipient cells taking allele with it
-there are potential problems with using viruses as gene delivery agents:
-viruses still may provoke immune or inflammatory response
-patient may become immune to virus, making subsequent deliveries difficult
-virus may insert allele into patient genome in location that disrupts gene involved in regulating cell division, increasing cancer risk
-virus may insert allele into patients genome in location that disrupts regulation of expression of other genes
Artificial chromosomes
-research done for possibility of inserting genes into artificial chromosome that would co-exist with other 46 chromosomes in target cell
-e.g. bacterial AC, yeast AC, and human AC
Germ line gene therapy
-germ line therapy involves altering genome of gametes or zygotes
-not only will all cells of that individual be altered, their offspring may also inherit the foreign allele
-this type of therapy has potential to change genetic makeup of many people, descendants of original patient, non of whom would have given consent
-also concerns about how genes may inserted- may find their way into a location that could disrupt expression or regulation of other genes or increase risk of cancer
-strict guidelines drawn up by regulatory bodies and ethic committees consider germline therapy for humans to be ethically impossible
Compare DNA and RNA
DNA
-double stranded, double helix
-anti parallel strands
-nucleotides added from 5’ end
-nucleotide (monomer) consists of phosphate group, pentose sugar (deoxyribose) and a nitrogenous base
-phosphodiester bonds between sugar and phosphate form sugar phosphate backbone
-C-G with 3 hydrogen bonds
-A-T with 2 hydrogen bonds
-nitrogenous bases= adenine and guanine are purines and cytosine and thymine are pyrimidines
-purines and pyrimidines are complementary base pairs
RNA
-polynucleotide, single stranded
-has phosphate, ribose sugar and nitrogenous bases
-however T is replaced with Uracil
-3 types: mRNA in transcription, tRNA in translation and rRNA in translation at ribosome
Describe process of DNA replication
-occurs in nucleus
-DNA unwinds using gyrase
-DNA unzips using helicase- hydrogen bonds between complementary base pairs are broken
-activated (by 2 phosphates from ATP) free nucleotides
-start at 5’ end, activated free nucleotides bind to complementary base pair on original strand
-A to T with 2 hydrogen bonds
-G to C with 3 hydrogen bonds
-occurs in 5’ to 3’ direction
-DNA polymerase catalyses addition of nucleotides
-phosphodiester bonds form sugar phosphate backbone
-known as semi conservative replication as consists of 50% original DNA and 50% ‘new’ nucleotides
Leading strand v lagging strand
-leading strand= synthesised continuously
-lagging strand= synthesised in fragments, discontinuously
-fragments joined by DNA ligase
Describe the incorrect models of DNA replication
CONSERVATIVE
-original molecule acts as template and new molecule forms
DISPERSIVE
-original DNA double helix breaks apart into fragments, and each fragment then serves as a template for a new DNA fragment
-as a result, every cell division produces two cells with varying amounts of old and new DNA
Describe the Meselson and Stahl experiment
-bacteria are grown in a broth containing the heavy (15N) nitrogen isotope
-DNA contains nitrogen in its bases
-as the bacteria replicated, they used nitrogen from the broth to make new DNA nucleotides
-after some time, the culture of bacteria had DNA containing only heavy (15N) nitrogen
A sample of DNA from the 15N culture of bacteria was extracted and spun in a centrifuge
-this showed that the DNA containing the heavy nitrogen settled near the bottom of the centrifuge tube
-the bacteria containing only 15N DNA was then taken out of the 15N broth and added to a broth containing only the lighter 14N nitrogen. The bacteria were left for enough time for one round of DNA replication to occur before their DNA was extracted and spun in a centrifuge
-If conservative DNA replication had occurred, the original template DNA molecules would only contain the heavier nitrogen and would settle at the bottom of the tube, whilst the new DNA molecules would only contain the lighter nitrogen and would settle at the top of the tube
-If semi-conservative replication had occurred, all the DNA molecules would now contain both the heavy 15N and light 14N nitrogen and would therefore settle in the middle of the tube (one strand of each DNA molecule would be from the original DNA containing the heavier nitrogen and the other (new) strand would be made using only the lighter nitrogen
How does Meselson and Stahl experiment prove semi conservative replication
-Meselson and Stahl confirmed that the bacterial DNA had undergone semi-conservative replication.
-The DNA from second round of centrifugation settled in the middle of the tube, showing that each DNA molecule contained a mixture of the heavier and lighter nitrogen isotopes
Polymerase chain reaction mixture
consists of:
-original DNA
-primers
-Taq DNA polymerase
-DNA free nucleotides
-magnesium ions- used as cofactors
How is the number of copies of DNA in PCR calculated
2^n
n= number of cycles
How is the number of cycles in PCR calculated
log2 (DNA copies)
Pyrosequencing
-uses sequencing by synthesis
-when correct nucleotide added, light is released
RP: electrophoresis
-DNA samples digested with restriction enzymes to cut them at specific recognition sites into fragments
-carried out at 35-40C
-agarose gel has buffer solution added to it
-loading dye added to tubes containing digested DNA
-digested DNA plus loading gel added to wells in electrophoresis gel using pipette- be careful not to pierce bottom of well in agarose
-connect to 18V battery and turn on
-leave for 6-8hours, a higher voltage can be used to shorten time
-DNA fragments move from cathode to anode due to their negatively charged phosphate groups
-the shorter fragments travel a further distance in a set amount of time
-pour buffer solution away and dye is added to gel to adhere the to DNA and stain the fragments
Define genetic engineering
-number of different processes to obtain a specific gene and place it in another organism
Describe reverse transcription
-mRNA is obtained during the transcription process
-reverse transcriptase enzymes uses mRNA as a template to make single stranded complementary DNA (cDNA)
-DNA polymerase makes single stranded cDNA to double strand of DNA
Issues of using pig insulin
-its not an exact copy of human insulin, therefore may not be as effective
-the immune system may also recognise it as non self and therefor break it down, triggering an immune response
-ethical issues of pig welfare, moral objections
-insulin must be purified and sterilised- makes process complex and costly
SCID
-severe combine immunodeficiency
-inability to produce an enzyme leads to accumulation of toxins that cause toxic death of T-lymphocytes
-ex vivo form of somatic therapy (cell removed, treated and replaced) meant genetically modified retrovirus contains ADA genes to synthesise the enzyme and no death of T lymphocytes
Pre implantation screening
-eggs fertilised by IVF, resulting embryos can be screened for genetic disorders, those without genetic disorders inserted into mothers uterus
-cost effective as no future treatment required
-better quality of life
Advantages and disadvantages of genetic screening
ADVANTAGES
-can identify presence of a disorder
-removes uncertainty- less anxiety
-allows early treatment
-preimplantation screening means only those without genetic disorders can be inserted into mothers uterus- cost effective as no treatment needed later and gives offspring better quality of life
-informs people about the choices they can make for healthy family planning
DISADVANTAGES
-false positives/ negatives
-may not be able to test for all mutations/ genetic disorders
-presence may not result in genetic disorder
-confirmed presence may cause stress and anxiety on parents
-discrimination against genetic disorders- designer babies and eugenics
-ethics against termination
