Molecular Techniques - DNA Flashcards
Which technique is used to amplify DNA?
Polymerase Chain Reaction
What components are required for PCR?
- Thermostable DNA polymerase (Taq) - synthesises new strands of DNA
- A pair of (specific) primers (forward and reverse) - uniquely define the region to be copied, starting point for DPol elongation
- Free dNTPs - to build new DNA strands
Describe the steps of PCR.
Consists of 20-40 temperature cycles, each involving 3 steps:
- Denaturing (95 C)
- Disruption of hydrogen bonds between complementary base pairs - forms ssDNA. - Annealing (50-65 C)
- Annealing of primers to complementary DNA regions.
- DPol binds to primer-template hybrid and begins polymerisation. - Polymerising (72 C)
- DPol synthesises a complementary DNA strand in 5’-3’ direction.
What are the applications of PCR?
- Restriction analysis
- Gene cloning
- DNA hybridisation and southern/northern blotting
- Reverse transcriptase-PCR
What is RT-PCR?
PCR variant used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcript from RNA.
Describe the process of RT-PCR.
- A long T primer is made which binds to the mRNA poly A tail.
- Reverse transcriptase then forms an RNA-cDNA duplex.
- The RNA is broken down to have a ssDNA copy of the RNA.
- This template DNA is amplified using normal PCR.
Which techniques are used to analyse DNA at the nucleotide level?
- Restriction analysis + gel electrophoresis
2. DNA sequencing
Which enzymes are required to perform restriction analysis?
Restriction endonucleases = bacterially-produced enzymes which degrade foreign DNA by recognising and cutting specific DNA sequences (restriction sites) - mostly palindromes of 4, 5, 6 or 8 bp.
What is the principle behind gel electrophoresis?
- DNA is negatively charged and will move towards the positive electrode (anode) if placed in an electric field.
- Smaller DNA fragments move faster than larger ones - DNA separated on the basis of size.
What are the requirements for restriction analysis/gel electrophoresis?
- Gel - matrix that allows DNA fragment separation
- Buffer - allows charge on the DNA samples across the gel
- Power supply - generates charge difference across the gel
- Stain/detection - to identify presence of separated DNA (e.g. Ethium bromide)
What are the applications of restriction analysis (+ PCR)?
- Investigate DNA fragment size, e.g. Small deletion
- Investigate mutations, e.g. Sickle cell disease
- Investigate DNA variation, e.g. DNA fingerprinting
- DNA cloning
Which molecules does the Sanger chain termination method (dideoxy chain termination method) of DNA sequencing use?
- Uses modified nucleotides: dideoxynucleotide triphosphate (ddNTP) molecules - similar to typical dNTPs but contain H rather than OH at 3’ position.
- Means that although it can be used by DPol as a substrate, it will block further elongation if incorporated into a growing DNA strand - no further phosphodiester bonds will be formed with a subsequent dNTP.
Describe the Sanger chain termination method (dideoxy chain termination method) of DNA sequencing.
- 4 separate tubes are used, each with a unique ddNTP and a labelled primer to initiate DNA synthesis.
- After incubation, reaction products undergo gel electrophoresis - separates labelled fragments out on basis of size.
- Read out sequence from bottom of gel to work out nucleotide sequence in newly synthesised strand.
How has the Sanger method of DNA sequencing been modernised?
- Now use fluorescently labelled ddNTPs all together in the same tube.
- Results can be produced as a chromatography from which we can read the sequence directly.
Which methods can be used to analyse DNA at the gene level?
- Gene cloning
- DNA hybridisation + southern/northern blotting
- RT-PCR
- Microarrays
- DNA fingerprinting/DNA profiling