Reverse Transcription & PCR (RT-PCR)

Question 1

what is transcription ?

Answer 1

The process of synthesising an RNA transcript with the transfer of sequence information from a DNA template

Question 2

what is DNA transcribed into ?

Answer 2

a complementary sequence of bases, called messenger ribonucleic acid (mRNA), which is then used for protein synthesis

Question 3

what is DNA transcribed into ?

Answer 3

a complementary sequence of bases, called messenger ribonucleic acid (mRNA), which is then used for protein synthesis

Question 4

The genetic code defines the relationship between…..

Answer 4

• the sequence of bases in the DNA (or RNA transcript)
• the sequence of amino acids in a protein

Question 5

The genetic code defines the relationship between…..

Answer 5

• the sequence of bases in the DNA (or RNA transcript)
• the sequence of amino acids in a protein

Question 5

The genetic code defines the relationship between…..

Answer 5

• the sequence of bases in the DNA (or RNA transcript)
• the sequence of amino acids in a protein

Question 5

The genetic code defines the relationship between…..

Answer 5

• the sequence of bases in the DNA (or RNA transcript)
• the sequence of amino acids in a protein

Question 6

What happens to DNA in reverese transcription ?

Answer 6

A copy of DNA from mRNA is made called cDNA

Question 6

What happens to DNA in reverese transcription ?

Answer 6

A copy of DNA from mRNA is made called cDNA

Question 6

What happens to DNA in reverese transcription ?

Answer 6

A copy of DNA from mRNA is made called cDNA

Question 7

What happens to DNA in reverese transcription ?

Answer 7

A copy of DNA from mRNA is made called cDNA

Question 8

what does PCR stand for ?

Answer 8

Polymerase Chain Reaction

Question 9

what does PCR do ?

Answer 9

Method used to amplify a sequence of DNA using a pair of oligonucleotide primers, each complementary to one end of the DNA target sequence

Question 9

what does PCR do ?

Answer 9

Method used to amplify a sequence of DNA using a pair of oligonucleotide primers, each complementary to one end of the DNA target sequence

Question 10

Essential Components of PCR – DNA Template

Answer 10

• Template DNA that contains target sequence
• Genomic (gDNA) or Complementary DNA (cDNA) can be used
• ≤ 1µg of DNA is required

Question 10

Essential Components of PCR – DNA Template

Answer 10

• Template DNA that contains target sequence
• Genomic (gDNA) or Complementary DNA (cDNA) can be used
• ≤ 1µg of DNA is required

Question 11

Essential Components of PCR – DNA Polymerase

Answer 11

1. A thermostable DNA Polymerase to catalyse template dependent synthesis of DNA
2. Taq polymerase is the enzyme of choice
3. Synthesise 9000 bases in < 10 seconds
4. Taq lacks 5′ → 3′ proofreading function
5. Misincorporation rate of 1 nucleotide in ≈ 9000 nucleotides

Question 11

Essential Components of PCR – DNA Polymerase

Answer 11

1. A thermostable DNA Polymerase to catalyse template dependent synthesis of DNA
2. Taq polymerase is the enzyme of choice
3. Synthesise 9000 bases in < 10 seconds
4. Taq lacks 5′ → 3′ proofreading function
5. Misincorporation rate of 1 nucleotide in ≈ 9000 nucleotides

Question 12

Principles of PCR

Answer 12

1. The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses
2. PCR amplifies a specific region of a DNA strand
3. amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size

Question 13

Essential Components of PCR – Primers

Answer 13

• Pair of synthetic oligonucleotides to prime DNA synthesis
• Complementary to the 3‘ ends of each of the sense and anti-sense strands of the DNA target
• DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers there is no double-stranded initiation site at which the polymerase can bind

Question 14

Essential Components of PCR – dNTPs

Answer 14

1. dNTPs – Deoxynucleoside triphosphates
2. Standard PCR contain equimolar amounts of dATP, dTTP, dGTP and dCTP
3. In a PCR reaction containing Taq and 1.5 mM MgCl2 [dNTP] 200-250 µM for each dNTP
4. In a 50 µl reaction this [dNTP] should allow synthesis of ≈ 6 – 6.5 µg DNA

Question 15

Essential Components of PCR – Buffer Solution

Answer 15

1. Buffer solution to maintain pH
2. Tris-HCl pH between 8.3 and 8.8 at room temperature
3. When incubated at 72°C pH of the reaction mixture drops to around 7.2

Question 16

Essential Components of PCR – Divalent Cations

Answer 16

1. All thermostable DNA polymerases require free divalent cations to:
   - React with dNTPs to form complexes that are substrates for the Taq polymerase
   - Stabilise the primer-template complexes

Question 16

Essential Components of PCR – Divalent Cations

Answer 16

1. All thermostable DNA polymerases require free divalent cations to:
   - React with dNTPs to form complexes that are substrates for the Taq polymerase
   - Stabilise the primer-template complexes

Question 17

Essential Components of PCR – Monovalent Cations

Answer 17

• Standard PCR buffer contains 50 mM KCl
• Works well for amplification of segments of DNA > 500 bp in length
• Increasing [KCl] to ≈ 70 – 100 mM may improve yield of shorter DNA fragments

Question 18

Essential Components of PCR – Monovalent Cations

Answer 18

• Standard PCR buffer contains 50 mM KCl
• Works well for amplification of segments of DNA > 500 bp in length
• Increasing [KCl] to ≈ 70 – 100 mM may improve yield of shorter DNA fragments

Question 19

PCR Procedure

Answer 19

1. Denaturation
2. Annealing
3. Extension/elongation
4. Final Extension
5. Hold

Question 19

PCR Procedure

Answer 19

1. Denaturation
2. Annealing
3. Extension/elongation
4. Final Extension
5. Hold

Question 20

Denaturing

Answer 20

1. First step in the cycling stage and consists of heating the reaction chamber to 94–98 °C for 20 – 30 seconds

This causes denaturation of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules

Question 21

Annealing

Answer 21

reaction temperature is lowered to 50–65 °C for 20 – 40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates

A typical annealing temperature is about 3–5 °C below the Tm of the primers used

During this step, the polymerase binds to the primer-template hybrid and begins DNA formation

Question 22

Extension

Answer 22

The temperature at this step depends on the DNA polymerase used; for Taq polymerase a temperature of 72 °C is commonly used

The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify

Usually between 30 – 60 seconds

Question 23

PCR Procedure – Number of Cycles

Answer 23

1. The processes of denaturation, annealing and elongation constitute a single cycle
2. Multiple cycles are required to amplify the DNA target to millions of copies
3. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles
4. A reaction set for 30 cycles results in 2 to the power of 30, copies of the original double-stranded DNA target region