lectures 2,3,4 - PCR, types of PCR and clinical uses

Question 1

how many cycles of PCR are completed?

Answer 1

at least 30

Question 2

what is the template for PCR?

Answer 2

double stranded DNA

Question 3

what are the primers in PCR?

Answer 3

small, ss DNA molecules, 6-30 bases long

Question 4

which end of the primer does DNA polymerase extend from?

Answer 4

3’

Question 5

what does DNA polymerase copy?

Answer 5

copies template DNA strand

Question 6

what do dNTPs stand for?

Answer 6

deoxyribonucleotide triphosphates

Question 7

magnesium is used in PCR. what is it a co factor for?

Answer 7

polymerase enzyme

Question 8

what is the purpose of magnesium in PCR?

Answer 8

to enhance the enzymatic activity of DNA polymerase

Question 9

what is the function of a buffer in PCR? what pH does it maintain?

Answer 9

maintains pH at 8-9.5

Question 10

what happens if primers are:

too long
too short

Answer 10

if too long, they won’t be specific
if too short, they will hybridise too slowly

Question 11

primers must have what percentage GC content?

Answer 11

40-60%

Question 12

which base pairs must primers end with?

Answer 12

GC pairs

Question 13

the 3’ end of primers are complementary to what?

Answer 13

the template DNA

Question 14

primer pairs must not have ……………. within themselves?

Answer 14

complementary regions

Question 15

taq polymerase is used when?

Answer 15

for repeated cycles

Question 16

if there is a band in the negative control lane of the GE (done from the PCR) what does it tell you about your DNA sample?

Answer 16

that it is contaminated and not pure

Question 17

what does the DNA molecular ladder in GE allow the user to estimate?

what is the unit of measurement?

Answer 17

the length of amplified DNA fragments

nucleotide bp

Question 18

what does reverse transcriptase PCR convert?

Answer 18

mRNA/RNA to cDNA

Question 19

which type of PCR is used to measure viral load of HIV so that the response to drugs can be monitored?

Answer 19

reverse transcriptase PCR

Question 20

what is the Ct value?

what does a lower Ct value indicate?

Answer 20

cycle threshold value = the cycle number at which the fluorescence reaches a threshold value

a lower Ct means that there is more cDNA in the starting template ( because fluorescence reaches threshold more quickly)

Question 21

at which phase of bacterial growth does real time PCR measure?

Answer 21

exponential phase

Question 22

at which phase of bacterial growth does end point PCR measure?

Answer 22

plateau phase

Question 23

which type of PCR requires reference genes such as Beta-actin, albumin and TATA box

Answer 23

qPCR

Question 24

which type of PCR uses fluorescent labels?

name the labels

Answer 24

qPCR - SYBR green and Taq man

Question 25

name the 2 PCR based techniques used for genotyping

Answer 25

PCR-FLP, ARMS-PCR

Question 26

name a pathogen for which PCR can be used to genotype it

Answer 26

tuberculosis

Question 27

which PCR based technique for genotyping uses a restriction enzyme?

Answer 27

PCR-FLP

Question 28

which disease can FLP-PCR be used to genotype?

Answer 28

Sorsby’s fundus dystrophy

Question 29

which disease can ARMS-PCR be used to genotype?

Answer 29

cystic fibrosis

Question 30

which type of PCR based technology used for genotyping diseases uses allele specific primers?

Answer 30

ARMS-PCR

Question 31

what type of bond do restriction endonucleases cleave?

Answer 31

phosphodiester bond

Question 32

what do restriction enzymes cleave? where?

Answer 32

cleave DNA at a specific site called a restriction site

Question 33

where are restriction enzymes naturally found?

what are they used for therefore?

Answer 33

found in bacteria

used to cut DNA that has been injected into them by viruses, to prevent the viruses reproducing inside of them

Question 34

give an example of a bacterial restriction enzyme, used to cut DNA at a GAATTC site

what does this cut create on the site?

Answer 34

EcoR1

creates sticky ends

Question 35

name biological lysing agents used to isolate DNA

Answer 35

sappanin for eukaryotes and lysozymes for bacteria (detergents)

Question 36

name the chemical/physical methods for lysing, used to isolate DNA?

Answer 36

chem - osmotic pressure to burst cells
phys - freeze thaw

Question 37

which two steps are done to purify isolated DNA?

Answer 37

use phenolchloroform then centrifugation

Question 38

on agarose gel, small DNA fragments move to which end?

why do they do this?

Answer 38

move to PLUS end

DNA is negatively charged and smaller DNA fragments move faster/greater distances than larger fragments

Question 39

on a graph which determines the size of DNA fragments, generated from GE, what would go on the X and Y axes?

Answer 39

on Y axis = (log10) DNA size (bp)
on X axis = distance migrated by band (mm) or Rf

Question 40

When designing primers for a standard single product PCR reaction what are the
constraints and considerations? What added complications come for multiplex PCR

Answer 40

primer length, melting temp, GC content, avoiding self-complimentarity, avoiding primer-dimer formation

complications for multiplex PCR: primer specificity, product size variation, cross contamination, primer design challenges

Question 41

At what stage of infection would you expect the lowest ct value?

Answer 41

early stage of infection