PCR

Question 1

purpose of PCR

Answer 1

make billions of copies of a specific DNA fragment or gene
AMPLIFICATION: PCR amplicons double with every cycle.

Question 2

overall PCR method: what goes into the test tube?

four things

Answer 2

1. DNA polymerase
2. template DNA
3. dNTPs
4. Forward and reverse primers

Question 3

what is the role of DNA polymerase for PCR?

Answer 3

DNA polymerase is the enzyme that makes new strands of DNA using existing strands as templates.

Question 4

What type of DNA polymerase is best for PCR? common type

Answer 4

thermally stable!
eg. Taq polymerase

Question 5

Mechanism of how DNA polymerase makes new strands of DNA

Answer 5

DNA polymerase requires PRIMERS as a starting point.
extends the primers in the 5’ to 3’ direction !! (IMPORTANT)
works at around 100-150 bp/s

Question 6

what are dNTPs, why are they needed for PCR?

Answer 6

deoxynucleotide triphosphates, carrying the base pairs –> essential building blocks for new DNA strands!
Acts as substrates for DNA polymerase to extend primers.

Question 7

main differences between different polymerases

Answer 7

error rate, proof reading etc
“better” polymerases are more expensive

Question 8

what is the key way of controlling PCR?

Answer 8

the primers are crucial for the specificity of the PCR, only the desired region is amplified.
affected by annealing temperature

Question 9

explain PCR method

Answer 9

three water baths:
1. 94˚C, 30s. DNA strands separate.
2. 50˚C, 30s. primers anneal.
3. 72˚C, 1 min. extension by polymerase.
REPEAT 25-30 times

Question 10

what is site-directed mutagenesis?

Answer 10

a technique used to introduce specific, intentional changes to the DNA sequence of a gene.
eg.
substitution of one nucleotide for another
insertion or deletion of nucleotides

Question 11

how is site-directed mutagenesis (SDM) achieved through PCR?

Answer 11

LONG PRIMERS! 30-50 mers, with BASES CHANGED toward the middle.
can make multiple changes at once
Changes are validated by sequencing (carried forward for copies)

Question 12

what is the purpose of adding restriction enzyme recognition sequences to primers? how does this change the design of the primer?

Answer 12

so that the DNA produced can be easily inserted into a plasmid vector.

RESTRICTION SITES added to the 5’ ends of primers (so they end up on the ends of the DNA copies). these sequences are recognised by restriction enzymes (eg. EcoRI, HindIII)

Question 13

Define ORF

Answer 13

open reading frame:
Starts with a start codon, ends with a stop codon, no stop codons between. encodes the whole protein without interruptions.
ie. DOES NOT CONTAIN INTRONS / UNTRANSLATED REGIONS (UTRs)! contains ONLY the coding sequence!

Question 14

why can eukaryotic genes not be directly cloned in bacteria? how can we get around this?

Answer 14

eukaryotic genes contain introns (non-coding sequences) and bacteria cannot process these (no splicing machinery).

Use cDNA in PCR, which then gets inserted into the plasmid.

Question 15

Define cDNA

Answer 15

complementary DNA
a mature DNA transcript that contains only the open reading frame, no introns.

Question 16

how is cDNA produced?

Answer 16

cDNA is transcribed from specific mRNA by using reverse transcriptase.

Question 17

how is PCR used in diagnostics?

Answer 17

can detect even tiny amounts of DNA or RNA from pathogens / mutagens / specific biomarkers
(amplified for detection)

high specificity achieved by the use of specific primers

Question 18

Why is PCR often used for cloning? what is the main difference compared to traditional molecular cloning previously?

Answer 18

non-restriction enzyme requiring technique
often faster, more flexible and seamless.

Question 19

define TA cloning.

Answer 19

a technique that allows insertion of a PCR-amplified DNA fragment into a plasmid vector WITHOUT the use of restriction enzymes.

(basically the simplest example of PCR based cloning - not really used)

Question 20

What is the mechanism by which TA cloning happens?

Answer 20

relies on the presence of single-A (adenine) overhangs on the PCR product, and T (thymine) overhangs on the plasmid vector.

Taq polymerase adds an A to the 3’ end of every copy. DNA ligase to insert into the plasmid.
NON-DIRECTIONAL insertion. but fast

Question 21

What is Gibson Assembly? How is PCR involved?

Answer 21

a PCR based cloning technique for seamless cloning
can be used to make a synthetic gene, or a plasmid with insert

DNA fragments with overlapping ends -PCR used to generate these by designing primers with homologous ends

Question 22

Gibson assembly reaction mixture contains:

Answer 22

1. T5 exonuclease to create single-stranded 3’ overhangs by “chewing back” the 5’ end
2. Phusion polymerase for extension
3. Taq ligase to seal

combine with DNA fragments (can have multiple) and incubate at 50˚C

Question 23

Gibson assembly mechanism

Answer 23

Each DNA fragment is generated with forward and reverse primers using PCR.

Exonuclease then digests the 5’ end, leaving complementary 3’ overhangs which anneal. DNA polymerase extends the 3’ ends and ligase seals the backbone nicks for a seamless join.