lectures 2,3,4 - PCR, types of PCR and clinical uses Flashcards
how many cycles of PCR are completed?
at least 30
what is the template for PCR?
double stranded DNA
what are the primers in PCR?
small, ss DNA molecules, 6-30 bases long
which end of the primer does DNA polymerase extend from?
3’
what does DNA polymerase copy?
copies template DNA strand
what do dNTPs stand for?
deoxyribonucleotide triphosphates
magnesium is used in PCR. what is it a co factor for?
polymerase enzyme
what is the purpose of magnesium in PCR?
to enhance the enzymatic activity of DNA polymerase
what is the function of a buffer in PCR? what pH does it maintain?
maintains pH at 8-9.5
what happens if primers are:
too long
too short
if too long, they won’t be specific
if too short, they will hybridise too slowly
primers must have what percentage GC content?
40-60%
which base pairs must primers end with?
GC pairs
the 3’ end of primers are complementary to what?
the template DNA
primer pairs must not have ……………. within themselves?
complementary regions
taq polymerase is used when?
for repeated cycles
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?
that it is contaminated and not pure
what does the DNA molecular ladder in GE allow the user to estimate?
what is the unit of measurement?
the length of amplified DNA fragments
nucleotide bp
what does reverse transcriptase PCR convert?
mRNA/RNA to cDNA
which type of PCR is used to measure viral load of HIV so that the response to drugs can be monitored?
reverse transcriptase PCR
what is the Ct value?
what does a lower Ct value indicate?
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)
at which phase of bacterial growth does real time PCR measure?
exponential phase
at which phase of bacterial growth does end point PCR measure?
plateau phase
which type of PCR requires reference genes such as Beta-actin, albumin and TATA box
qPCR
which type of PCR uses fluorescent labels?
name the labels
qPCR - SYBR green and Taq man
name the 2 PCR based techniques used for genotyping
PCR-FLP, ARMS-PCR
name a pathogen for which PCR can be used to genotype it
tuberculosis
which PCR based technique for genotyping uses a restriction enzyme?
PCR-FLP
which disease can FLP-PCR be used to genotype?
Sorsby’s fundus dystrophy
which disease can ARMS-PCR be used to genotype?
cystic fibrosis
which type of PCR based technology used for genotyping diseases uses allele specific primers?
ARMS-PCR
what type of bond do restriction endonucleases cleave?
phosphodiester bond
what do restriction enzymes cleave? where?
cleave DNA at a specific site called a restriction site
where are restriction enzymes naturally found?
what are they used for therefore?
found in bacteria
used to cut DNA that has been injected into them by viruses, to prevent the viruses reproducing inside of them
give an example of a bacterial restriction enzyme, used to cut DNA at a GAATTC site
what does this cut create on the site?
EcoR1
creates sticky ends
name biological lysing agents used to isolate DNA
sappanin for eukaryotes and lysozymes for bacteria (detergents)
name the chemical/physical methods for lysing, used to isolate DNA?
chem - osmotic pressure to burst cells
phys - freeze thaw
which two steps are done to purify isolated DNA?
use phenolchloroform then centrifugation
on agarose gel, small DNA fragments move to which end?
why do they do this?
move to PLUS end
DNA is negatively charged and smaller DNA fragments move faster/greater distances than larger fragments
on a graph which determines the size of DNA fragments, generated from GE, what would go on the X and Y axes?
on Y axis = (log10) DNA size (bp)
on X axis = distance migrated by band (mm) or Rf
When designing primers for a standard single product PCR reaction what are the
constraints and considerations? What added complications come for multiplex PCR
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
At what stage of infection would you expect the lowest ct value?
early stage of infection
