Practical 9: PCR Flashcards Preview

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Flashcards in Practical 9: PCR Deck (22)
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1
Q

where was enzyme Taq DNA polymerase isolated from

A

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

2
Q

what should you find in a PCR reaction tube

A

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

3
Q

where does PCr take place and what is the first stage

A

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

4
Q

second step of PCR

A

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

5
Q

how do we stop the template strands from reannealing

A

add the primers in excess to outcompete other annealing events

6
Q

what is the third step of PCR

A

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

7
Q

what is the role of the primers

A

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

8
Q

what are primers

A

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

9
Q

how does the amplicon form

A

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

10
Q

what is genotyping

A

determining the genotype of ann individual

11
Q

what are SNPS

A

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

12
Q

what are SNTRs

A

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

13
Q

what are VNTRs

A

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

14
Q

what are transposons

A

jumping genes. interspersed genome wide repeats

15
Q

what is phenylthiocarbamide / PTC

A

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

16
Q

what is TAS2R38

A

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

17
Q

Taster T and non taster t, allels differ how

A

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

18
Q
if someones genotype was 
TT
Tt
tt
what would their corresponding phenotype be
A

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

19
Q

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

A

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

20
Q

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

A

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

21
Q

what is PCR-RFLP

A

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

22
Q

enzymes that cleave DNA are called

A

restriction endonucleases