PCR & NGS Flashcards
Describe Reverse Transcription
- mRNA can be extracted
- in the cell mRNA goes on to be translated to form the final protein the gene encodes
- in reverse transcription a DNA copy (cDNA) of the mRNA is made
- this cDNA can then be used for downstream applications
What doe PCR stand for ?
Polymerase Chain Reaction
What is PCR ?
A method used to amplify a sequence of DNA using a pair of oligonucleotide primers, each complememtary to 1 end of the DNA target sequence
What are the principles of PCR ?
- It amplifies a specific region of DNA
- the amount of amplified product is determined by the available substrates in the reaction
What are the essential components of PCR ?
- DNA template
- DNA polymerase
- primers
- dNTPs
- Buffer solution
- Divalent cations
- monovalent cations
Describe the DNA template part of PCR?
- template DNA which contains the target sequence
- genomic or complementary DNA can be used
Explain the DNA polymerase part of PCR
- A thermostable DNA polymerase catalyses template dependent synthesis of DNA
- Taq polymerase is the enzyme of Choice
Explain the primer aspect of PCR
- Pair of synthetic oligonucleotides to prime DNA synthesis
- complementary to 3’ ends of each of the sense and anti-sense strands of the DNA target
Detail the role of dNTPs of PCR
- dNTPs = Deoxynucleotide Triphosphates
- standard PCR contain equimolar amounts of dATP, dTTP, dGTP & dCTP
Describe the role of buffers in PCR
- maintains pH
- Tris-HCL pH between 8.3 - 8.8 at room temperature
Describe the role of Diavalent Cations of PCR
- thermostable DNA polymerases require free divalent cations - Mg2+ Mn2+
- reacting with dNTPs to form complexes that are substrates for the Taq polymerase
- stabilises the primer-template complexes
What are the different stages of PCR ?
- Denaturation
- Annealing
- Extension/Elongation
- Final Extension
- Hold
How many cycles are there in the denaturation, annealing & elongation stages ?
20-40 cycles per step
Describe the 1st step of PCR –> Denaturing
- heating the reaction chamber to 94-98 degrees for 20-30 seconds
- causes denaturation of the double-stranded DNA template by breaking hydrogen bonds between bases, yielding 2 single-stranded DNA molecules
Describe the 2nd step of PCR –> Annealing
- temp is lowered to 50-65 degrees for 20-40 seconds
- allows annealing of the primers to each end of the single-stranded DNA templates
- efficiency & specificity of annealing is strongly affected by annealing temperature (Ta)
- polymerase binds to the primer-template hybrid & begins DNA formation
Describe the 3rd step of PCR –> Extension
- temp depends of the DNA polymerase
- for Taq polymerase = 72 degrees
- new DNA strand is synthesised, which is complementary to template strand by adding free dNTPs
- usually lasts between 30-60 seconds
Describe the 4th step of final extension/hold
- only optional step
- performed at 70-74 degrees for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated
- hold step cools the reaction chamber to 4 degrees for an indefinite time
What does NGS stand for ?
Next Generation Sequencing
What is transcriptomics ?
- study of the transcriptome = complete set of RNA transcripts that are produced by genome
What are the main 2 transcriptomic techniques ?
- Microarrays
- RNA-Seq
Describe Microarrays
- measures the abundances of a defined set of transcripts via hybridisation
- allows assay of thousands of transcripts simultaneously
- usually consists of a grid of short nucleotide oligomers known as probes
Define RNA-Seq
- accomplished by reverse transcribing RNA in vitro & sequencing resulting cDNA
- Input RNA amounts are much lower for RNA-Seq compared to microarrays - allows for finer examination
What are some applications of transcriptomics ?
- diagnostic & disease profiling
- gene function annotation
- responses to environment
- non-coding RNA
