Manipulating Genomes Flashcards
What types of genes are used for DNA profiling, and why?
Introns because there will be some differences in base sequences in exons as they code for characteristics like eye colour
What is satellite DNA and the two types?
- Within introns
- Short, repeated DNA sequences
- Always appear on the same area of chromosomes
Minisatellite - 20 to 50 base pairs long, repeated 50-100 times (aka VNTR’s)
Microsatellite - 2-4 base pairs, repeated 5-15 times (STR’s)
What is the basic/essential principle of DNA profiling?
- Different people will have a different number of repeats
- Therefore they will generate a different satellite pattern
What are the stages of DNA profiling?
- Extract DNA and amplify it by putting it through PCR to replicate the same DNA
- Digestion
- Separation, carry out gel electrophoresis
- Southern blotting carried out, where the DNA fragments are transferred onto a membrane
- Hybridisation, add florescent dye and put under UV light, to visualise evidence
Explain the stage of digestion in DNA profiling
Add enzyme restriction endonucleases, which recognise specific DNA sequences (restriction sites) and cut up the DNA into smaller fragments whilst leaving satellites intact
Explain gel electrophoresis
- Using electricity to separate DNA fragments
- DNA fragments placed in wells on agar jelly on negative side of electrode, so negative DNA repels away and towards anode
- Agar gel is immersed alkali to separate DNA double strands into single strands
- DNA fragments separated by length, but can’t be seen until dye is added
- Smaller fragments move through the gel faster
Briefly explain the 3 steps of PCR
Denaturation (95 degrees) - separating the double helix as H bonds are broken
Annealing (55) - primers bind to the start of the target DNA
Synthesis (72) - building up the new strand
What are primers?
- Short DNA sequences that bind to the start of a gene you want to amplify
- Designed to have complementary bases to gene
Explain the stage of synthesis in PCR
- 72 degrees
- Free nucleotides pair up to exposed bases by complementary base pairing
- Taq polymerase joins up backbone of new strand to form phosphodiester bonds
- Taq because human DNA polymerase will denature at 72 degrees
What is DNA sequencing and the 5 ingredients required?
- Technique where we can map out the base sequence of an individual’s genome
Need -
1. DNA sample to be sequenced
2. Free nucleotides (in excess)
3. Coloured fluorescent labelled terminator bases
4. DNA polymerase
5. DNA primers
Explain the stages of DNA sequencing
- Heat up to 95-96 degrees to denature DNA and separate the double helix and form 2 separate strands
- Cool down to 50 degrees so the primers anneal to the single strands
- Heat up to 60 degrees, DNA polymerase starts to join up paired nucleotides to synthesise new strand
- Once terminator base binds, strand can no longer extend
- Ends up with lots of strands of different lengths
- Because free nucelotides are in excess, there will be less terminator bases than nucleotides, all possible DNA chains will be produced
Explain terminator bases
- Have a hydrogen atom instead of a hydroxyl group at the 3 prime end of the ribose ring
- Therefore they cannot form phosphodiester bonds with the next nucleotide
- Sequence will end
What are the 2 ways we can read the data from DNA sequencing?
- Do electrophoresis and southern blotting
- Terminator bases are all labeled, so direct UV light onto membrane
or - more recent way using laser over electrophoresis
- generates pattern on computer
Define bioinformatics and computational biology
Bioinformatics - Development of software and computing tools needed to organise and analyse raw biological data, includes development of algorithms, mathematical models and statistical tests
Computational biology - uses data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances
What is synthetic biology?
The design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems
What were the issues with using insulin from pigs?
- Ensuring it was properly sterilised
- Religion
Outline the process of genetic engineering of insulin in bacteria
- DNA extracted from human cell and desired gene identified using a process such as DNA sequencing
- Plasmid extracted from bacteria as they contain a marker gene
- Restriction endonuclease cuts out section where desired gene is, and same enzymes used to cut out marker gene from plasmid
- Marker gene and desired gene bind on recombinant plasmid
- Place recombinant plasmid in bacteria, which becomes transgenic as it carries a gene from a different type of organism
- Bacterium cultured in agar plate
What is a marker gene?
A gene used to determine if a nucleic acid sequence has been successfully inserted into an organisms DNA
Explain the main method of isolating a gene in genetic engineering
Using restriction endonucleases - enzymes that recognise specific restriction site on genome, and cut it out
- They can either do one clean cut through the DNA sequence, or do a staggered cut
- Staggered cut produces sticky ends, which have exposed bases so can easily bind to plasmid
What is the less common method of isolating a gene in genetic engineering?
Using reverse transcriptase
- Allow DNA to be first transcribed, leaving mRNA of desired gene
- Extract mRNA from cytoplasm, and mix with reverse transcriptase, to form complementary DNA (cDNA)
- Put cDNA into plasmid
Explain how to insert the desired gene into the vector in genetic engineering
- Extract plasmid from bacteria, or use a virus
- Cut plasmid at the marker gene using the same restriction enzyme that was used to isolate the gene, to produce the same sticky ends
- Blue marker gene produces blue pigment, but can’t be transcribed once desired gene is inserted into it as it’s cut in half, so can’t produce blue pigment
- DNA ligase forms phosphodiester bonds between desired gene and plasmid/marker gene
- Other marker gene, ampicillin resistant gene, so can survive in plate with ampicillin
- Recombinant DNA formed
What is the difference between DNA ligase and DNA polymerase
- DNA ligase joins DNA fragments together at a single point
- DNA polymerase joins individual DNA nucleotides together working across a whole template strand
Explain the principle of transformation in genetic engineering
- Recombinant plasmid needs to be placed into host cell (bacteria)
- To make cell membrane more permeable, so plasmid more likely to be taken in
Explain the three methods of transformation in genetic engineering
Method A
- Put plasmid and bacteria in calcium rich solution, and do a heat shock to create pores in membrane
Method B
- Electroporation, creates pores in membrane using electricity
- Small current passed through
Method C
- Electrofusion
- Have plasmid inside a vesicle and provide electric current through that and bacterial cell
- Causes them to assimilate and fuse together and plasmid passes through from vesicle to bacteria
What are the 3 possibilities of bacteria at the end of genetic engineering, and what would happen when they’re all mass produced together?
1 - Bacteria didn’t take up plasmid, so bacteria remains the same
- Doesn’t have ampicillin resistant gene, so dies and won’t appear on plate
2 - Bacteria does take up plasmid, but plasmid was an original plasmid without desired gene
- Will grow and survive, and produce blue pigment
3 - Successful, bacteria took up plasmid, and plasmid was recombinant containing desired gene
- Will survive and grow, but won’t produce blue pigment
What happens in mass production after genetic engineering?
- Culturing of bacteria in agar gel with nutrients and ampicillin
Explain the process of genetic engineering in plants
- Uses agrobacterium tumefaciens, a bacterium that causes tumours in healthy plants
- A desired gene, like pesticide production is placed in the Ti plasmid of A. tumefaciens, along with a marker gene like antibiotic resistance or flourescance
- This is carried directly into the plant cells DNA
- The transgenic plant cells form a callus, which is a mass of gentically modified plant cells, each of which can be grown into a new transgenic plant
