Section 8 - In-vivo and In-vitro cloning etc. (until end) Flashcards
What does in-vivo mean?
In life
What does in-vitro mean?
In glass
What is the importance of sticky ends?
DNA from different sources can be joined together if they have the same sticky end
How can sticky ends be joined together?
Using DNA ligase
Joins the sugar-phosphate backbone together
What is the new DNA molecule combing sticky ends and DNA called?
Recombinant DNA
What is a plamid?
Used to transport DNA into a host cell
What are plasmids?
Most commonly used vector
Why are plasmids useful?
Nearly always contain antibiotic resistant genes
What is the role of the plasmid in in-vivo cloning?
Contains 2 antibiotic resistance genes
One is disrupted when restriction enzymes cut open the plasmid
Other used to select cells which have taken up the plasmids
What is the process of in-vivo cloning?
- DNA is cut using restriction enzyme
- Same restriction enzyme is used to cut open the plasmid
- Enzyme is denatured
- Plasmid now has sticky ends complementary to donor DNA
- DNA ligase joins 2 DNAs together
- Recombinant DNA is now created
What is transformation in in-vivo cloning?
Insertion of DNA into host cell
Plasmids must be reintroduced into host cell
Bacteria, plasmids and Ca2+ ions are mixed together
How is a bacteria containing the plasmid identified?
Growing bacteria in a medium that contains an antibiotic
Found in the plasmid and bacteria will only survive in the presence of an antibiotic if the genes are present and functioning
How is bacteria containing plasmid with DNA fragments identified?
Gene markers are used to identify which plasmids have taken up the DNA fragment
Gene marker is disrupted if the DNA fragment is present
What can gene markers be?
Resistant to an antibiotic
Fluorescent protein
Enzyme whose action can be identified
Why is replica plating used?
Used to identify the bacteria with the recombinant plasmid
Bacteria are transferred to identical positions on plates containing ampicillin and then tetracycline
Bacteria with the donor DN will be able to grow on ampicillin, but not tetracycline
Why are gene markers necessary during in vivo gene cloning?
Gene markers are used to identify which bacterial plasmids have taken up DNA fragments
What is an advantage of using fluorescent gene markers rather than antibiotic gene markers?
Not killing bacteria you want
Easy to identify
What are some of the arguments for and against the use of DNA technologies?
For:
Microorganisms modified to produce large range of substances –> used to treat diseases
GM plants crops can help to prevent certain diseases
GM crops can be engineered to have financial and environmental advantages
Against:
Impossible to predict accuracy
GM bacteria often have antibiotic resistance marker genes that have been added
Long-term consequences cannot be identified
Ethical considerations
What is transfection?
Inserting the correct gene into a cell
Only suitable for disease caused by a single gene
What is somatic (body cell) cell therapy?
Copies of the correct gene are inserted directly into the somatic of the sufferers
Doesn’t prevent the disease from occurring in the next generation
Has to be repeated many times as the effects don’t last very long
What is germ line therapy?
Corrected gene is inserted into a fertilised egg produced IVF
If successful, all cells of the embryo contain the corrected gene due to mitosis
It is permanent and would ensure offspring inherit corrected gene
(illegal in UK)
What are the 2 main vectors?
Viruses
Liposomes
How can viruses be used at vectors?
- Adenovirus modified to make them harmless
- New gene is taken up by adenovirus. Gene becomes part of the virus DNA
- Viruses isolated and sprayed into nostrils of patients
- Viral DNA (including new gene) inserts into epithelial cells of patients lungs
What are the advantages of viruses in cell therapy?
Efficient at entering cells
Can be targeted to specific cells
Easy to modify to prevent damage to cell
What are the disadvantages of viruses in cell therapy?
Only suitable for small amounts of genetic material
Can cause an immune response
May not insert into host DNA so short lived
May insert incorrectly disrupting other genes
How are liposomes used in gene therapy?
The gene within a plasmid is packaged within small spheres of lipid bilayer
What are the advantages and disadvantages of using liposomes in cell therapy?
Can carry larger genes
Less likely to trigger an immune response
Less efficient if modified plasmid DNA doesn’t integrate with host DNA
How do you know where a DNA sequence is located?
DNA probes (locate specific alleles of genes)
DNA hybridisation
What are DNA probes?
short, single stranded DNA with a label attached to it so they’re easily identified
Complementary to a section of DNA under investigation
What are the 2 ways DNA probes can be labelled?
Radioactive marker - revealed by photographic film
Fluorescent marker - emits a colour on exposure to UV light
Why are probes small?
As long ones may partially anneal to a variety of sequences and bind in several places
No use in determining a very specific sequence of bases required
What is the method of locating a specific allele of a gene?
- Determine sequence of nucleotides on mutated allele using DNA sequence techniques or genetic libraries
- Make a DNA fragment with a complementary base sequence to the mutated allele
- Use PCR to make lots of copies of DNA probe
- Obtain DNA from person with mutated allele and heat it to separate 2 strands
- Cool separated strands and mix with DNA probes
- If mutated allele is present the DNA probe will bind to the complementary base sequence
- Wash DNA to remove unattached probes
- Remaining hybridised DNA will be labelled with fluorescent marker
- Detect with UV light and special microscope
How is a DNA microarray used?
- Fluorescently label a human DNA fragment
- Wash this over the array
- If any human DNA sequence match any of the DNA probes sequences - they will stick to the array
- Wash the array and remove unattached DNA
- Expose to UV light
Any spot that fluoresces has that specific mutant allele
What are the uses of DNA probes?
Locating specific, desired gene for genetic engineering
Identifying same gene on a variety of genomes from separate species - for comparison studies of genomes
Identifying presence of an allele for a disease
DNA probes are used to:
Help identify inherited conditions
Help determine how patients will respond to specific drugs
Help identify health risks
What is genetic counselling?
Advising people about screening - looking for mutated allele if in family history and explaining results of screening
Enables people to make personal decisions about themselves and their offspring
What does genetic counselling involve?
Researching family history of an inherited disease
Advising parents of likelihood of it arising in children
Informs effects of a disease
What is genetic fingerprinting?
Technique used by scientists which is based on the fact that DNA of every individual, except identical twins is unique
What are variable number tandem repeats (UNTRs)?
Regions found in the non-coding part of DNA
Short sequence is repeated a variable number of times at a single location (satellite DNA)
How is genetic fingerprinting done?
DNA extracted from sample
If sample is small it is amplified by PCR
- Restriction enzymes are used to cut the DNA of different base sequences close to the VNTR regions
- Distance between recognition sites will change depending on the number of repeated sequences
- DNA fragments of different lengths are produced from different DNA samples
- Electrophoresis of the fragments will produce a distinctive pattern
What are the uses of genetic fingerprinting?
Forensic scientists - identify suspects of crimes (profile compared to suspects)
Forensics to identify unidentifiable body parts
Individuals at risk of developing particular diseases
Familial relationships for paternity or immigration cases
Species conservation to reduce chances of inbreeding in captive breeding programmes
What can scientists insert into a plasmid to ensure only wanted gene is expressed ?
Promoter region
How can a scientist insert copies of a gene into a plasmid?
Plasmid cut open using restriction endonuclease, leaves complementary sticky ends
DNA ligase used to join complementary sticky ends
How many copies of each original DNA molecule would be present after 5 cycles of PCR?
32
How would you know if someone tested positive for a having a certain allele on a gel electrophorysis?
Has the highest band that travelled the shortest distance so has largest number of repeats
How would scientists know whether the number of repeats they wanted was shown on a gel?
Run fragments of known length at the same time
What is the reason for only one band being present on a person?
Homozygous
Fragments are the same length
Suggest and explain why a drug that combines gene therapy and RNA interference (siRNA) is more effective than either treatment alone? [4]
siRNA ensures mRNA from mutant gene isn’t translated into defective protein
Gene therapy means normal copy of gene can be inserted into muscle cell, restoring function
Only RNA = no functioning protein
Only gene therapy = defective protein stops cell functioning
What is meant by gene therapy?
Introduction of healthy gene - replacement of defective gene
When a modified nucleotide is used to form a complementary DNA strand, the sequencing reaction is terminated. Suggest how this sequencing reaction is terminated?
Does not form a bond or react with other nucleotides
Doesn’t fit enzyme active site
