PCR & NGS

Question 1

Describe Reverse Transcription

Answer 1

• 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

Question 2

What doe PCR stand for ?

Answer 2

Polymerase Chain Reaction

Question 3

What is PCR ?

Answer 3

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

Question 4

What are the principles of PCR ?

Answer 4

• It amplifies a specific region of DNA
• the amount of amplified product is determined by the available substrates in the reaction

Question 5

What are the essential components of PCR ?

Answer 5

• DNA template
• DNA polymerase
• primers
• dNTPs
• Buffer solution
• Divalent cations
• monovalent cations

Question 6

Describe the DNA template part of PCR?

Answer 6

• template DNA which contains the target sequence
• genomic or complementary DNA can be used

Question 7

Explain the DNA polymerase part of PCR

Answer 7

• A thermostable DNA polymerase catalyses template dependent synthesis of DNA
• Taq polymerase is the enzyme of Choice

Question 8

Explain the primer aspect of PCR

Answer 8

• 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

Question 9

Detail the role of dNTPs of PCR

Answer 9

• dNTPs = Deoxynucleotide Triphosphates
• standard PCR contain equimolar amounts of dATP, dTTP, dGTP & dCTP

Question 10

Describe the role of buffers in PCR

Answer 10

• maintains pH
• Tris-HCL pH between 8.3 - 8.8 at room temperature

Question 11

Describe the role of Diavalent Cations of PCR

Answer 11

• 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

Question 13

What are the different stages of PCR ?

Answer 13

• Denaturation
• Annealing
• Extension/Elongation
• Final Extension
• Hold

Question 14

How many cycles are there in the denaturation, annealing & elongation stages ?

Answer 14

20-40 cycles per step

Question 15

Describe the 1st step of PCR –> Denaturing

Answer 15

• 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

Question 16

Describe the 2nd step of PCR –> Annealing

Answer 16

• 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

Question 17

Describe the 3rd step of PCR –> Extension

Answer 17

• 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

Question 18

Describe the 4th step of final extension/hold

Answer 18

• 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

Question 19

What does NGS stand for ?

Answer 19

Next Generation Sequencing

Question 20

What is transcriptomics ?

Answer 20

• study of the transcriptome = complete set of RNA transcripts that are produced by genome

Question 21

What are the main 2 transcriptomic techniques ?

Answer 21

1. Microarrays
2. RNA-Seq

Question 22

Describe Microarrays

Answer 22

• 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

Question 23

Define RNA-Seq

Answer 23

• 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

Question 24

What are some applications of transcriptomics ?

Answer 24

• diagnostic & disease profiling
• gene function annotation
• responses to environment
• non-coding RNA