6.3: Manipulating genomes Flashcards
What is the development of sequencing
- Sanger sequencing
- DNA sequencing machine using fluorescent dyes
- High throughput sequencing
What is the sanger technique?
5 steps
Used for gene sequencing
- Extract DNA, cut it into fragments of various lengths
- Amplify those lengths using pCR
- Sequence the DNA by adding it to 4 different solutions
DNA nucleotides
DNA polymerase
Primers
A terminator base - Add them onto an electrophoresis plate which separates out DNA based upon mass
- Read the seuqence of DNA which can be stored on a database
What is the first DNA sequencing machine and what did it do?
Based on Sanger’s method
Fluorescent dyes instead of radioactivity were used to label the terminator bases
These dyes glowed when scanned with a laser beam and the light signature was identified by computer
What is high throughput sequencing?
Fast, cheap methods to sequence genomes
What are applications of gene sequencing?
5
- The human genome project
- Comparing between species
- Variation between individuals
- Evolutionary relationships
- Predicting the amino acid sequence of proteins
What is the human genome project?
When it was launched, in 2003, it sequenced the human genome and found only 24000 genes
What does comparing the genome between the species do?
Allows us to find out what is conserved
Many of the differences between organisms are not because the organisms have totally different genes but because some stored genes have been altered and work in different ways
- Some changes to regulatory regions of DNA that do not code directly for protein have also altered the expression of the genomes
What does variation between individuals’ genomes show?
- Humans are genetically similar except for rare cases where gene has been lost by deletion of a part of a chromosome
- Same genes but different alleles
- SNPs are places on the DNA where subsitutions (mutations) can occur
- Methylation
What is methylation?
Adding a CH3 to the DNA which alters the gene expression
Prevents transcription as the alteration means RNA polymerase cannot bind to the gene
How can gene sequencing predict the amino acid sequences of proteins?
Use the genome sequence and researchers knowledge of which genes code for a specific protein
Due to their knowledge of the base triplets which code for the amino acid
They can find out the primary structure of proteins
What has sequencing allowed for?
- Synthetic biology
- Bioinformatics
- Epidemiology
- Protemics
What is synthetic biology?
Field of science that involves redesigning organsims for useful purposes by engineering them to have new abilities
To solve problems in medicine, manufacturing and agricultures
What is protemics?
Large scale study of a set of proteins produced in an organism, system or biological context
What is bioinformatics?
Software is developed to process and understand large complex data (DNA sequences) using computational biology
- Allows access to large amount of data
- Information is stored as universal
- Allows rapid comparison of sequences with newly sequenced alleles
- Amino acid sequences/protein structures held database
- Computer modelling of new protein structure from base sequences
What is epidemiology?
Bioinformatics can be used in it
The study of how often diseases occur in different groups of peole are why
- Identify source of outbreak
- Identify vulnerable populations
- Design vaccination programs to target certain individuals
What happens in DNA profiling?
7 steps
- DNA is extracted
- DNA is amplified in the process of PCR
- Cut DNA using restriction enzymes
- DNA is put into gel electrophoresis plate which separates the DNA based on the mass
- A banding pattern can be seen
- The DNA to which the individual is being compared is treated with the same restriction enzymes and also subjected to electrophoresis
- The banding patterns of the DNA samples can then be compared
What types of DNA is used in DNA profiling?
Short tandem repeats (STR) - small regiion 2-4bp repated 5-15 times
Variable number tandem repeates (VNTR) - 20-50bp repeated 50-several 100 times
Why are introns used in genetic profiles?
- In most people genome is very similar
- Using coding sequences of DNA would not provide a unique profile
- Non - coding DNA contains VNTR/STR/repeating sequencing
What are uses of DNA profiling?
- Paternity test to discover the father of a child
- Suspects at a crime scene
- Searching for genes that can trigger diseases
What is PCR (polymerase chain reaction)?
DNA is obtained from a crime scene from a criminal and it is amplified enabling it ot be analysed
What does PCR rely on?
3x
- DNA is made of two antiparallel backbone strands
- Each strand has a 5’ end and a 3’ end (only grows from the 3’ end)
- Base pairs pair up according to complementary base pairings
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)
- Requires the addition of primer molecules to make the process start
- A cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to replicated
What is the process of PCR?
8 steps
- The sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq polymerase
- Mixture is heated to around 94-96 to break hydrogen bonds between base pairs - denaturing double stranded DNA
- Mixture is cooled to 68 so primers can anneal to one end of each single stranded DNA. Gives a small section of double-stranded DNA at the end of each
- Taq polymerase enzyme molecules can now bind to the end where there is double stranded DNA
- Temp raised to 72 which keeps the DNA as single strands
- Taq polymerase catalyses the addition of nucleotides to the single stranded DNA molecules starting at the end with the primer and going in 5’ to 3’
- Taq reaches end of the DNA molecule, then a new DNA has been generated
- Whole porcess begins again and is repeated for many cycles
What is electrophoresis?
Used to separate different sized fragments of DNA for identification and analysis
What occurs in electrophoresis when separating fragments?
Technique occurs in agarose gel covered by buffer solution
- Pipette the 4 different terminator bases into the different wells
- Pass a current through the electrophoresis plate from cathode to anode
- DNA has a slightly negative charge due to lots of phosphate groups so it will be repelled by the cathode and attracted to the anode
- Smaller fragments will travel further as they have less mass and therefore has less resistance in the gel
What are the two ways to see the DNA after electropherosis?
Southern blotting - using radiactive probes and x rays
GFP (green fluorescent protein) - using DNA probe and UV light
How is electrophoresis used in separating proteins?
Same as DNA fragments but carrieed out in presence of charged detergent-sodium doceyl sulfate (SDS)
Equalises the surface charges on the molecules and allows proteins to separate as they move through the gel
According to molecular mass
What is conditons is electrophoresis used to diagnose from the analysis of types of haemoglobin?
4x
- Sickle cell anemia
- Aplastic anemia
- Thalanaemia
- Leukemia
What is a DNA probe?
Short single stranded length of DNA that is complementary to a section of the DNA being investigated
What two ways are DNA probes labelled using?
A radioactive marker - once probe has annealed by complementary base pairing to the piece of DNA, it can be revealed by exposure to photographic film
A fluorescent marker - emits a colour on exposure to UV light
Why are DNA probes used for locating specific DNA sequences?
- To locate a specific gene needed for use in genetic engineering
- To identify the same gene in a variety of different genomes from different species when conducting genome comparison studies
- Identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition
What are microarrays?
Fixed surface that number of different probes are placed on
MICROARRAYS
What happens in genetic engineering?
6 steps
- Restriction enzymes are used to cut the desired gene from DNA
- Creates sticky ends which make it easier to insert desired genes
- The plasmid (vector) is cut using the same restriction enzymes to produce complementary sticky ends
- The desired gene is inserted into the vector/plasmid using an enzyme called DNA ligase. Inserted with a marker
- The recombinant DNA is inserted into the host cell using electroporation. This is where an electric shock makes the membrane porous therefore the plasmids can pass through the membranes of the host cell
- The host cell undergoes mitosis, reproducing the deisred gene too. This is then tested to ensure the desired gene has been taken up by the host cell by looking at fluorescence or applying antibiotic
What is reverse transcription?
Makes DNA from an RNA template
Does the opposite of transcription
What is recombinant DNA?
DNA combined from 2 sources
What is the vector used for
- Plant
- Bacteria
- Animal
- Agrobacterium tumefaciens, plasmids, virus
- BAC (bacteria artificial chromosome), bacteriophage, virus
- Virus, BAC, plasmid
What are positive and negative issues of using microorganisms in genetic manipulation?
Positive:
- Make insulin to treat diabetes
- Growth hormone
Negative:
- Escape to wild and transfer markers genes for antibiotic resistance
What are positive and negative issues of using mice in genetic manipulation?
Ethical issue
Positive:
- Medical research
- Used to develop therapies for breast and prostate cancer
Negative:
- Some people object the use of animals
What are positive and negative issues of using pharmaceutical proteins in genetic manipulation?
Ethical issue
Positive:
- Genes for human pharmaceutical proteins to treat hereditary emphysema, can be inserted into goats or sheep
- Human protein they express into they milk is harvested
Negative:
- Concerns for the welfare of the sheep and the goats
What are positive and negative issues of using crop plants resistant to pests in genetic manipulation?
Positive:
- Do not need to use pesticides
- Better for environment
Negative:
- Concerns farmers fo not want GM seeds and would not have the choice to buy non GM seeds
business concerns
What are positive and negative issues of using golden rice in genetic manipulation?
Positive:
- GM to contain beta carotene that can be present in the rice grains
Negative:
- Concerns farmers would have to buy the seed every year
What is gene therapy?
Insert a functional allele of a particular gene into cells that contain only mutated and non functioning alleles of that gene
If the inserted alle is expressed, then the individual will produce a functioning protein and no longer have the symptoms associated with the genetics disorder
What are the two types of gene therapy?
Somatic cell therapy
Germ line therapy
What is somatic cell gene therapy?
Gene therapy by inserting functional alleles into body cells
- Affects only certain cell types
- The alterations made to the genome in those cells are not passed to the patients offspring
How are lipsomes used for gene therapy?
Made up of alleles which are packaged within small spheres of lipid bilayer
- Placed into aerosol inhaler and sprayed into noses of patients
- Some pass through plasma membrane of cells lining the respiratory tract pass through the nuclear envelope and insert into the host genome
- The host cell will express teh CFTR protein (CF patients lack)
in vivo
How are viruses used for gene therapy?
Genetically modified so that it encases the functioning allele to be inserted into the patient and being made unable to cause a disease
Enters the recipient cell taking the allele with it
What is germ line gene therapy ?
Gene therapy by inserting functional alleles into gametes/zygotes
- Offspring may also inherit the foreign allele
- Potential to change the genetic makecup of many people
What are issues with gene therapy?
Virus:
- May still cause an immune or inflammatory response in the patient
- Patient may become immune to the virus making subsequent deliveries difficult/impossible
Insertions:
- Genes find their way into a location that could disrupt the expression or regulation of other genes or increase the risk of cancer
Liposomes:
- Only really work in respiratory and low transfection efficiency
