Molecular Techniques Flashcards
Restriction enzyme
Enzyme that recognises and binds to a specific 4-6 base pair DNA sequence called a restriction site as its active site complementary to the DNA sequence.
Enzyme breaks phosphodiester bonds on specific positions on both DNA strands, creating blunt or sticky ends depending on the type of RE.
Used as a defense mechanism by bacteria against bacteriophages by cleaving foreign DNA, hence restricting multiplication of viruses.
Gel electrophoresis
Genomic DNA is extracted from soft tissue/dried blood of organism and digested with same restriction enzyme)
Dense loading buffer is mixed with DNA sample to help it sink to bottom of wells in agarose gel located near the cathode. Loading dyes are added to DNA sample to allow visualisation of progress of electrophoresis.
Buffer contains ions that allow conduction of electric current.
Negatively-charged DNA migrates out of well towards anode when subjected to electric current. Meshwork of agarose polysaccharides impede progress of longer fragments more than shorter ones, causing them to migrate slower and end up nearer to well.
Method of visualisation
If sufficient DNA fragments of discrete sizes
Stain gel with ethidium bromide followed by visualisation under UV light
If insufficient
Southern blotting followed by nucleic acid hybridisation with radioactive DNA probes which will hybridise DNA by complementary base pairing
Using autoradiography with an X-ray film, banding pattern can be visualised
Role of loading dye
Contains glycerol to make DNA sample denser so can sink to bottom of well
Colours invisible DNA to check for correct loading
2 coloured dyes act as visual markers for to show progress of migration of DNA fragments in gel
One typically runs ahead of the DNA sample and another runs after sample
PCR
Heating to 95deg separates two strands of DNA double helix by breaking hydrogen bonds between complementary bases, denaturing DNA into single strands.
Cool to around 64deg with excess primer allows primers to anneal specifically to regions flanking the target DNA sequence via complementary base pairing, providing 3’OH end for chain extension.
At 72deg, optimum temperature for Taq polymerase which performs chain extension by synthesising complementary DNA strand from 3’ end of primer by complementary base pairing.
Taq polymerase why obtained from genetically modified E. Coli
- Allows for mass production of Taq DNA polymerase
- Hot spring bacteria may require specific conditions (high temp and nutrients) for synthesis while E. Coli can be easily cultured at low temp
Limitations of PCR
- Taq polymerase lacks 3’ to 5’ proofreading ability
Errors occurring early in PCR reaction will get compounded with each replication cycle and all resulting daughter molecules will be exponentially affected - Synthesis of PCR primers requires knowledge of sequences flanking target region to be amplified.
If unknown, no proper primers can be synthesised. - Limited size of DNA fragment that can be amplified
Efficiency of amplification decreases with increasing size as polymerase tends to fall off template DNA during chain extension - Exponential amplification of contaminant DNA
Unwanted DNA sequences can be amplified to large amounts alongside target DNA sequences
What is RFLP
Restriction Fragment Length Polymorphism refers to unique banding pattern among individuals when the genomic DNA is digested by restriction enzymes and separated by gel electrophoresis.
General outline for detection of allele
not essay
- Extract genomic DNA from cells in blood/mouth swab
- Carry out PCR using primers complementary to nucleotide sequences flanking allele
- Cut amplified fragments using restriction enzyme
- Carry out gel electrophoresis on DNA fragments
- Carry out southern blotting and nucleic acid hybridisation using radioactive DNA probes complementary to allele
- Visualise using autoradiography on X-ray film and carry out RFLP analysis: compare banding pattern with banding patterns of known genotypes
RFLP and Sickle Cell Anaemia Theory
Sickle-cell anaemia is an autosomal recessive genetic disease caused by substitution mutation in DNA coding for B-globin in haemoglobin. Red blood cells have an abnormal, rigid sickle shape that can lead to anaemia, obstruction of blood capillaries due to sickling, organ damage and death.
Since mutation is located at a restriction site, the same restriction enzyme can no longer recognise the site. When normal and diseased alleles are digested by same restriction enzyme, they will produce different mixtures of fragments for each allele. We can thus infer the genotype of individuals by RFLP analysis.
How to do RFLP
To detect polymorphisms, DNA fragments are transferred to nitrocellulose membrane and made single-stranded using an alkaline solution.
Nucleic acid hybridisation is carried out by incubating the membrane with radioactive DNA probes complementary to region of interest. Radioactive probes bind to DNA fragments by complementary base pairing, appearing as bands in autoradiography with an X-ray film, highlighting the differences in restriction sites and thus DNA polymorphisms in individuals.
or
Conduct PCR with primers flanking boundaries of gene → digest w restriction enzyme → run gel electrophoresis
Visualise bands via ethidium bromide and UV light
If banding patterns are similar, likely to be from very closely-related individuals
Why does RFLP work
Due to presence of DNA polymorphisms in different individuals, there will be variations in the number and location of restriction sites, and and number of tandemly repeated nucleotide sequences.
What is RFLP used for
Disease detection
DNA fingerprinting in forensics/paternity testing
RFLP and Sickle Cell Anaemia Steps
To detect disease, DNA from affected individual is digested with restriction enzyme and digested DNA is separated by gel electrophoresis. Southern blotting is carried out where DNA is transferred to nitrocellulose membrane and made single-stranded using NaOH for nucleic acid hybridisation using radioactive DNA probes complementary to B-globin gene on either side of restriction site.
Since HbS has lost restriction site, it will yield a larger fragment as visualised via autoradiography with an X-ray film. Single large band: 2 HbS alleles Two bands (1 int 1 small): Normal HbAHbA Three bands (1 large 1 int 1 small) Normal HbAHbS
