6.3 - Manipulating genomes Flashcards
1
Q
Formation of recombinant bacteria e.g. bacteria that can produce insulin
A
- identify gene for insulin production
- isolate the gene using a restriction enzyme to leave sticky ends
- cut plasmid open using the same restriction enzyme to leave complementary sticky ends and mix with isolated genes
- Bases on complementary sticky ends complementary base pairs A-T and G-C with hydrogen bonds from between bases
- DNA ligament added to seal nicks in sugar phosphate backbone to form recombinant DNA
- Mix plasmids with E. Coli bacteria - encourage them to take up plasmids using heat shock and calcium salts (or electroporation) some take up bacteria
- identify the ones which have taken up plasmid by using replica plating
- provide bacteria with nutrients in a fermenter and to allow insulin to be made by the bacteria
- extract the insulin and purify in downstream processing
2
Q
Methods of forming recombinant bacteria to produce insulin before gene was located
A
- isolate mRNA for insulin (had not found gene yet)
- use reverse transcriptase enzyme to synthesise a single stranded complementary DNA strand
- Add DNA polymerase and DNA nucleotides to the single strands to produce a copy of the gene for insulin (cDNA)
- add unpaired nucleotides at the ends to give complementary sticky ends to the ones to be cut on the plasmid
- cut plasmid open using restriction enzymes and mix with cDNA genes
- Based on complementary sticky ends complementary base pairs A-T and C-G with hydrogen bonds form between the bases
- DNA ligament added to seal nicks in sugar phosphate backbones to form recombinant DNA
- Mix plasmids with E.coli bacteria - encourages them to take up plasmids using heat shock and calcium salts (or electroporation) some taken up by bacteria
- identify the ones which have taken up the plasmids using replica plating
- provide bacteria with nutrients in a fermenter and to allow insulin to be made by the bacteria
- extract the insulin and purify in downstream processing.
3
Q
Advantages of using insulin produced by genetically engineered bacteria
A
The old method of obtaining insulin was from pigs. Advantages over this method are:
- engineered insulin is cheaper (as foodstock is cheaper than that for pigs)
- much larger amount of product is more readily as the rate of production is much faster
- there is less risk of infection than with pig insulin
- avoids side effects / allergies / immune response that for some people experience with pig insulin
- ethically it is advantageous to use bacteria as there are no animals right issues associated with them as there are with pigs. This is also true for religious groups e.g. Jews who may not want pig insulin for religious reasons
4
Q
Using genetic markers in plasmids to identify bacteria that have taken up recombinant plasmid
A
- replicate plating - antibiotic resistance (gene) introduced and survivors have plasmid
- fluorescents markers (gene) introduced and glowing bacteria have plasmids
- identify bacteria producing insulin using antibiotics
5
Q
Vectors
A
- carries DNA from one cell to another
- transgenic animals - virus/BAC/liposome
- somatic gene therapy - virus/liposome
- genetically engineered plants - agronacterium tumefaciens / plasmid
- transgenic bacteria - plasmid /BAC
6
Q
Gene therapy
A
Delivery of new functional versions of genes into DNA of patients to treat genetic diseases
SOMATIC CELL GENE THERAPY
- augmentation - adding a functional version of a gene so that the correct protein is made in order to relieve symptoms
Done by:
- ex vivo - take cells out, modify the replace
- vectors - virus or liposome to deliver desired allele to cells (not very effective)
GERM LINE GENE THERAPY
- genetically engineered gametes, zygotes or early embryo
- all cells in new organism will have desired gene
- may pass on desired gene to offspring
- done in animals - Illegal in humans
7
Q
Why is it easier to perform gene therapy when normal allele is the dominant allele
A
- has effect when added to genome
- not masked
- no need to, remove / inactivate, recessive / mutant, allele
8
Q
Comparing somatic cell and Germ line gene therapy
A
SOMATIC CELL GENE THERAPY
- changes body cells
- can’t pass on new genes to offspring
- specialized cells are treated and don’t divide. Can’t pass on genes to other cells. Need to repeat gene therapy regularly (as specialized cells are replaced)
- legal in humans
- harder to deliver genes. Has to be ex vivo or in vectors (which can be ineffective)
GERMLINE GENE THERAPY
- changes gametes/zygotes/embryos
- can pass on new genes to offspring
- no need to repeat therapy as every cell and hence every new cell will contain a copy of new genes
- not legal in humans for ethical reasons - danger of designer babies/eugenics
- delivering gene is easier as it is straight into germ cells
9
Q
Benefits and concerns of somatic cell gene therapy
A
Benefits
- reduce symptoms and giver better quality of life - less need for medication
- can treat diseases such as cystic fibrosis
- extends lifespan
Concerns
- virus vectors may cause viral disease
- procedures may be painful
- temporary and so needs to be repeated
10
Q
Why germline gene therapy is illegal
A
- could (unintentionally) introduce an genetic disease
- Permanent changes to human DNA that can be passed on - considered unethical.
- Could lead to eugenics or ‘designer babies’
- patient has no say in DNA being modified
11
Q
Advantages and disadvantages of genetic screening
A
Advantages
- can identify presence of disorder
- removes uncertainty
- allows early treatment
- which may improve, life expectancy / quality of life
- allows informed choice about having children
- allows IVF and embryo screening
- allows fetal testing and termination
- choice, re donation / adoption
Disadvantages
- false, positives / negatives
- only small number tests available / not available for all conditions
- presence may not result in condition
- confirmed presence gives stress / fear
- problem re, telling / testing, rest of family
- discrimination by, employers / insurers
- ethics of termination
- could increase intolerance / discrimination, of disabled
12
Q
Creating clone libraries to map entire genome
A
- Genes are mapped to find which chromosome they are from
- Samples of the genome are cut with restriction enzymes to give fragments of 100,000 bp
in length - Each fragment is inserted into a separate bacterial artificial chromosome (BAC) (plasmids)
and each BAC is put a different E. coli cell. - The bacteria divide and each produce an individual clone library of their specific fragment.
To sequence the BAC fragments:
- The fragments of the genome are extracted from the plasmids.
- Fragments are cut up using different restriction enzymes - produces many fragments of
different lengths up to about 1000bp.
- Each piece of DNA is now sequenced using the ‘chain-termination method.’
- Finally, computers are able to put all of the fragments back in order to sequence the
genome as a whole.
13
Q
Polymerase Chain Reaction (PCR)
A
- Small fragment of DNA to be copied is mixed with DNA nucleotides, primers and Taq DNA
polymerase - Heated to 95oC – denaturation step
- Temperature breaks the H-bonds between the complementary base pairs in the DNA to
make 2 single strands of DNA. . - The temperature is cooled to 55oC. - annealing step.
- At this temperature, the primers will bind to each single strand of DNA at the 3’ end. This
allows the DNA polymerase to bind to the double stranded sections. - Temperature is increased to 72oC - elongation step
- Taq DNA polymerase adds DNA nucleotides to single strand according to base pairing
rules. This will eventually create a copy of the original fragment of DNA. - This process is repeated over an over again and the number copies of the DNA fragment
increases exponentially.
n.b Taq DNA polymerase comes from a thermophilic bacteria and so doesn’t denature at 95 degrees - its optimum is 72oC
14
Q
Advantages of PCR for copying DNA compared with copying DNA using BACs and visa versa
A
Advantages of PCR
- PCR quicker – only takes a few hours rather than weeks
- PCR uses less space – BACs require multiple agar plates
- PCR less labour-intensive – set up and left to run all done in one step
Advantages of BACs
- in vivo less expensive – PCR equipment and chemicals are expensive
- BACs less technically complex – conditions are not so critical
- Longer pieces of DNA can be cloned – PCR limits size of DNA fragment it is possible to
copy
15
Q
Electrophoresis
A
- DNA is cut into smaller fragments using restriction enzymes.
- The fragments are placed into the wells at the end of the gel plate where the negative
electrode will be - The plate is immersed into a tank filled with buffer solution and an electrical current is
passed through the tank - DNA is negatively charged and so is attracted to the other end of the plate, where the
positive electrode is, so the molecules diffuse along the gel to the other end - The shorter fragments move further in the same period of time than the longer ones
- The banding pattern is invisible so the DNA must be stained with ethidium bromide and
then viewed under UV light to observe the final banding pattern
