DNA Amplification by PCR Flashcards
basic principles of PCR
allows for exponential amplification of specific sequences of DNA for down steam applications
Xeroxing process
amplicon - amplified product, STR, etc
involves heating and cooling samples in a precise thermocycler
oligonucleotide primers determine the boundaries of the amplified product
these are complimentary to the 3’ ends of the sequence of the 3’ ends of the sequence of interest
used 2x in forensic str typing
qPCR
STR amplification
three differences
detector in qPCR
use fluorescent probe in qPCR
in STR amplification for typing, using capillary electrophoresis, primers are labeled with a dye, not the probe with a dye and quencher
thermocycler parameters
2 steps in qPCR remember annealing and extension combined so probe doesn’t fall off
3 steps in STR amplification
essential per components
thermostable dna polymerase
per primers
other components
dNTPs
template DNA
divalent cations such as Mg2+
buffer
both divalent cations and buffer maintain pH and salt level
thermostable DNA polymerase
thermos aquaticus (Taq) polymerase enzyme
discovered in the late 1980s greatly increased efficiency and allowed for automation
amplitaq gold DNA polymerase is most commonly used in forensic applications
also used in qPCR briefly mentioned earlier
new amplitaq gold 360
same enzymes just improved
PCR reactions are usually set up in RT which could result in nonspecific annealing between primers and template
annealing between primers, primer dimer
all this reduces amplification efficiency
minimized by hot start PCR
AmpliTaq is a modified enzyme
remains inactive in pH below 7
buffer used in a hot start is temp sensitive and when the temp is increased the pH increases
modifier comes off permanently
enzyme is activated by heating to 95C in addition to dna denaturation
inhibits enzyme activity at RT, upon activation the modifier is permanently released
all polymerases are not created the same
dna polymerase characteristics
initiation/regulation
how and where polymerization is activated and how its controlled
polymerization rate
number of nucleotides polymerized per second
error rate
number of incorrect nucleotides
processivity
number of nucleotides added per binding event (before polymerase falls off)
proofreading functions
does it have an error correcting function
primer specificity
ABI amplitaq Gold was found to be the gold standard for per polymerases in forensic kits
has a hot start modification for RT set up
optimal operating temp is 72, extension is at 72
used in qPCR which takes advantage of the 3’ endonuclease activity to eat up the probe, which emoves the quencher dye from the reported dye, removes anything in its way
polymerization rate = 60 sec/kb
processitivity <5kb
how many bases can it add before it doesn’t want to work anymore
per primers
oligonucleotides that are complimentary to the sequences that flank the target region of template
forward and reverse primers are req
req for exponential growth
primers much be specific to the target sequence or nonspecific products might interfere with interpretation
~15 nucleotides long
primer pairs should have similar melting Tm
primers should bot share similar sequences
primer dimer
attach to each other
Tm=melting temp
Tm=81.5+16.6(log[K+])+0.41(%[G+C])-(675/n)
k+ is the conc of potassium ions
G+C is the GC content (%) of the oligonucleotide
n is the number of bases of theoligonucleodide
CG/AT comp, CG has stronger bonds
higher n, higher Tm, more nucleotides to anneal
PCR parameters
cycles
denaturation
2 complimentary DNA template strands are separated at a high temp, breaking H bonds between nucleotides
annealing
temp decreased to allow the primers to find and teach anneal to their complimentary sequence on the dna template
usually 3-5C lower than the Tm of the oligonucleotide primer
extension
temp is increased tot he optimal operating temp for polymerase
taq is 72C
amplification controls
monitor the effectiveness of PCR amplification
amped along with samples using same reagent sand same batch in thermocycler
same consumables, plastic products, pipettes, not just reagents
positive control
shows the per componenets are working properly
reagents, thermal cycling parameters
use a standard DNA template
007
negative controls
controls for contamination from reagents and or consumables
reagent blanks are a negative control for extraction
neg amplification control is amped along with samples with no dna samples in it, just buffer and reagents
rfu
relative fluorescence unit
electropherogram
plot of amplicons
stochastic threshold
lowest amount of fluorescence observed where you are confident you are visualizing both alleles if hererozygote (have a low probability of dropout)
analytical threshold
user defined, an amount of fluorescence when a peak is considered an analytical signal and not noise
adenylation
non-template addition of +A the addition of usually an adenine by Taq polymerase on the 3’ end of the per product while copying template dna
let step of PCR
by adding A+ to all amplicons, all amplicons will be the sample length
removing this step results in a split peak (1bp apart)
quirk of Taq, does it sometimes not all
therefore we made it do it all the time
factors affecting PCR
degradation
observed in qPCR ratio of small vs large
longer targeted sequence, more likely chance of degradation, might have many small frags and few large frags
LCN - <50pg DNA
low copy number
inhibitors - observed in qPCR
contamination
template degradation
breakdown of dna into smaller fragments
longer the amplicon length, the more risk of breaking down
shorter amplicons less affected
if you have a degraded sample, you can try Minifiler which has shorter primers and therefore shorter amplicons
amplifies 8 largest loci
amplicons are shorter than 270 bp
x axis is time which is proportional to length
ski slope effect
shows degradation
y axis is RFU, relative fluor unit
all relative no unit
allele call, repeat number
loci, targeted sequences
x axis is time reflects amplicon length
more length, more time to get thru system
without degradation, peaks should be around same height
can still get a profile from degraded DNA (low ski slope)
low copy number of template
random not always the same
encountered when amplifying very low levels of DNA
phenomenon occurs which one of the two heterozygote allele fails to amplify
unequal sampling of the two alleles
leads to dropout and false interpretation of homozygous
stochastic effect
two alleles in a heterozygous individual are unequally detected at a low level of starting dna
ways to overcome drop out of alleles is to increase cycle number when amplifying samples
perform 3 separate amplifications, consensus profile
another way to address the issue when interpreting samples is to assign the alleles a p-anything for example
a small peak (below stochastic threshold) for allele 15 at vWa could be homozygous or could be any of the heterozygote pairs 13,15 14,15, 15,16, etc
STRmix doesn’t have stochastic one utilize p-anything
p-anything attributes to all possibilities
stochastic levels are determined in-house validations depends on instruments and methods
levels at you are confident that there is no drop out
