Practical 9: PCR

Question 1

where was enzyme Taq DNA polymerase isolated from

Answer 1

Thermus aquaticus, a bacteria that lives in high temperature steam vents

Question 2

what should you find in a PCR reaction tube

Answer 2

DNA templte containing the original sequence of DNA to be amplified
two single stranded primers which target specifically the fragment to amplify
Taq DNA polymerase
free nucleotides called dNTPs
reaction buffers

Question 3

where does PCr take place and what is the first stage

Answer 3

in the thermal cycler
denaturation at 90-95C.
the double stranded DNA melts open into single stranded DNA molecules

Question 4

second step of PCR

Answer 4

annealing around 50-60C (temperature dependent on primer sequences). drop in temperature allows primers to find their complementary sequences in the single stranded template and bind (anneal) through base complementarity

Question 5

how do we stop the template strands from reannealing

Answer 5

add the primers in excess to outcompete other annealing events

Question 6

what is the third step of PCR

Answer 6

extension at 72C for eg 30-60 sec per Kb to be amplified. ideal temp for taq polymerase to extend from the primer by added dNTPs complementary to the template one by one in the 5’ to 3’ direction

Question 7

what is the role of the primers

Answer 7

provide a sbort sequence of double stranded DNA for the Taq polymerase to extend from.

Question 8

what are primers

Answer 8

primers are short synthetic single stranded DNA molecules that are complementary to the beginning and ned of the DNA fragment to be amplified

Question 9

how does the amplicon form

Answer 9

after extension each single strand from the template has been replicated into double stranded DNA (dsDNA) which serves as a template in the next cycle. 1dsDNA becomes 2, 2 becomes 4 etc. by the end of 30 cycles one has 2^30 or over 1 billion copies (amplicon or PCR product) of the original target sequence as the template

Question 10

what is genotyping

Answer 10

determining the genotype of ann individual

Question 11

what are SNPS

Answer 11

short nucleotide polymorphisms, arise by errors in DNA replication and account for 90-95% of polymorphisms

Question 12

what are SNTRs

Answer 12

short tandem repeats. short DNA sequences of 2-7bp that are repeated up to 100 times. also called microsatellites

Question 13

what are VNTRs

Answer 13

variable number tandem repeats. longer DNA sequences of 15-200bp that are repeated up to 100 times. also called minisatellites

Question 14

what are transposons

Answer 14

jumping genes. interspersed genome wide repeats

Question 15

what is phenylthiocarbamide / PTC

Answer 15

a chemical compound that is tasted as either very bitter, mildly bitter, or tasteless

Question 16

what is TAS2R38

Answer 16

PTC tasting gene. codes for a taste receptor on the tongue

Question 17

Taster T and non taster t, allels differ how

Answer 17

by three single nucleotide polymorphisms at nucleotide positions 145, 785 and 886

Question 18

if someones genotype was 
TT
Tt
tt
what would their corresponding phenotype be

Answer 18

TT PTC tastes very bitter
Tt PTC tastes mildly bitter
tt PTC is tasteless

Question 19

Genotyping TAS2R38 alleles using PCR followed by RFLP takes advantage of what

Answer 19

the second SNP (C785T) disrupts the recognition site of the bacterial restriction enzyme and so generates a restriction fragment length polymorphism RFLP following digestion by this enzyme

Question 20

using RFLP how can we tell the difference between T and t

Answer 20

for T the enzyme can bind and cut so we get two fragments
for t the enzyme can no longer bind (as it doesn’t have that nucleotide sequence and therefore doesn’t have the recognition site ) and so will not cut, the PCR product will remain intact after digestion ie 1 fragment

Question 21

what is PCR-RFLP

Answer 21

first amplify by PCR the DNA locus that contains the SNP, and then perform digestion of the PCR product

Question 22

enzymes that cleave DNA are called

Answer 22

restriction endonucleases