Basic genetic engineering 3

Question 1

What is cDNA?

Answer 1

Double stranded DNA made by copying eukaryotic mRNA

Question 2

How is cDNA made?

Answer 2

mRNA is copied into single stranded DNA using a reverse transcriptase enzyme

Question 3

How is cDNA made double stranded?

Answer 3

The single stranded DNA is made double stranded using a standard DNA polymerase or a heat stable DNA polymerase

Question 4

Why is cDNA useful? (3)

Answer 4

• Copy of a gene which contains no introns
• cDNA can be used to make the protein product of the gene in a pro/eukaryotic system
• Bacteria lack introns and machinery to remove them

Question 5

Describe the construction of cDNA? (6)

Answer 5

1. Oligo(dT) primer pairs and copies 3’ poly(A) tail of mRNA strand
2. Synthesis of the strand occurs until viral reverse transcriptase come along - hairpin occurs
3. NaOH removes RNA strand
4. DNA polymerase primer extends from the 3’ cDNA hairpin end
5. S1 nuclease cuts at the hairpin
6. Double stranded cDNAS is formed

Question 6

What is PCR?

Answer 6

PCR is the amplification of a specific DNA sequence in vitro

Question 7

What are the molecule conditions for PCR? (3)

Answer 7

• Can be amplified from a very low or high level
• The DNA doesn’t have to be pure or clean
• Done in small tubes with 50ul, thin walled

Question 8

What does PCR require? (5)

Answer 8

• A sample of DNA
• 2 primers complementary to different strand of required DNA
• A buffer (provide suitable conditions)
• Building blocks of DNA (dGTP, dATP, dTTP, dCTP)
• Heat stable DNA polymerase

Question 9

What are the key concepts of PCR?

Answer 9

Repetitive heating 90C (DENATURE), cooling 60C (ANNEALING) and DNA synthesis 72*C (EXTENSION) amplify the DNA between primer building sites

Question 10

How many cycles of denaturing, annealing and extending in PCR are required?

Answer 10

20-30

Question 11

What are the characteristics of a PCR primer? (4)

Answer 11

Short
Single stranded
Oligonucleotides
Identical to different strand of required DNA

Question 12

What is primer 1 called in PCR and what is its role?

Answer 12

Primer 1 is called the forward primer

Has the same sequence as the top strand of the DNA

Question 13

What is primer 2 called in PCR and what is its role?

Answer 13

Primer 2 is called the reverse primer

Has the same sequence as the bottom strand of the DNA

Question 14

Which genes do Pol 1 and Pol 3 have?

a) 5’-3’ polymerase
b) 3’-5’ exonuclease
c) 5’-3’ exonuclease
d) polymerisation rate

Answer 14

. Pol 1 | Pol 3

a) Yes | Yes
b) Yes | Yes
c) Yes | No
d) 16-20 | 250-1000

Question 15

Are Pol 1 and Pol 3 suitable for PCR?

Answer 15

No - don’t work at high enough temperatures

Question 16

Which polymerase is used in PCR?

Answer 16

Taq polymerase

Question 17

What are the key features of Taq Polymerase? (2)

Answer 17

> Half life at 95*C is 1.6hrs

> Lacks a 3’-5’ exonuclease (makes errors)

Question 18

How does the double helix of DNA denature then renature? (3)

Answer 18

1. Heat up: starts to unwind
2. Keep heating: separates out
3. Cool down: back to start

Question 19

Explain the steps in the PCR: (4)

Answer 19

1. Heat reaction to 94*C - denature target DNA (30secs)
2. Rapidly cool reaction to 60*C - allows primers to base pair with complementary strands (annealing) (rapid)
3. Rapidly heat to 72*C to allow Taq polymerase to synthesise 2 new DNA strands with the primers (extension) (30secs)
4. repeat steps 1-3 20/30 times

Question 20

How much DNA does 1 PCR cycle produce and how long is 1 cycle?

Answer 20

Doubles the amount of DNA fragment

2 minutes

Question 21

How can we work out what DNA is once the PCR has finished?

Answer 21

Agarose gel

Question 22

What are the kay parts of a primer? (6)

Answer 22

• 17-30 nts long
• GC content 50%
• Annealing temp = [2(AT)+4(GC)]
• Avoid runs of a single nucleotide
• Avoid complementary sequences
• Include C or G at the 3’ end of each primer - stronger

Question 23

What is a restriction enzyme site?

Answer 23

An extra A nucleotide is added to the 3’ end of each DNA strand - easy to insert into a vector

Question 24

What are the application of PCR? (4)

Answer 24

Molecular analysis
Single cell analysis
Genome analysis
Clinical pathology