Lecture Two: Polymerase chain reaction and forensic DNA profiling (finished) Flashcards
what was PCR first used for?
to identify skeletal remains using HLA DQα locus
At first to use PCR on VNTRs the sample had to be what?
fresh
By the 1990s PCR was used for what?
was used to amplify STRs
whats another word for
Human minisatellites?
variable number tandem repeats
How many bp’s do variable number tandem repeats have?
6-100bp
Amplified Fragment length Polymorphisms tandem repeats have an amplicon size smaller than what?
smaller than 1 kb to allow successful amplification
Human microsatellites are also known as what?
short tandem repeats
what stem typical core repeat for a STR?
2-6bp
whats the amplicon size of a STR?
Amplicon size 100-500 bp.
whats a Dinucleotide repeat?
same two repeat, example : (CA)(CA)(CA)
whats a simple repeat?
Tandem repeats with identical repeat units.
what do Non-consensus Alleles (microvariants) contain?
Contain incomplete repeats by one or more nucleotides
whats a
Compound Repeat?
Consist of more than one type of simple repeat.
what does a Complex Repeat contain?
Contain several clusters of different tandem repeats with intervening sequences.
whats a simple definition for PCR?
Repeated copying of a selected region of a DNA molecule.
where is PCR done?
in vitro
who won the noble prize for PCR?
Kary Mullis in 1985
what are the 3 main steps of PCR?
Denaturation
Annealing
Extension
what happens at denaturation?
Denaturation at high
temperature (92°C-95°C)
what happens at annealing?
Annealing at a cooler annealing temperature (50°C-65°C)(~5°C below primers’ Tm; optimised experimentally)
what happens at extension ?
Extension at a temperature between the annealing and denaturing temperatures (usually at 72°C)
whats one cycle of PCR?
denature- heat to separate strands
annealing - hybridisation of primers
extension - DNA synthesis from primers
whats the average cycle length in PCR?
25-35 cycles
at what cycle is the correct size double stranded target created?
cycle 3
what is adenylation (A tailing)?
adding of a non-template nucleotide to the 3ʹ end.
what does incomplete adenylation show as?
this will appear as a split peak on the
electropherogram (epg) due to the different size PCR products.
what amplicon is favoured for adenylation?
+A amplicon
In adenylation how do you go from -A amplicon to +A amlicon?
by a final incubation step.
what are the three phases of a PCR amplification curve?
Exponential
Linear
Plateau
Till when does the exponential phase continue?
The exponential phase will continue until one or more of the components of the reaction become limited.
what does the linear phase in the PCR amplification curve show?
The linear phase is an indication that the amplification efficiency is decreasing.
what happens to the plateau phase in the PCR amplification curve?
The plateau phase is where no new amplicons are accumulating due to the exhaustion of reagents.
what are the 5 components of PCR?
- Template DNA
- Oligonucleotide primers
- DNA polymerase
- Deoxynucleoside triphosphates (dNTPs) • Buffer
what does a PCR commercial master mix contain?
- DNA polymerase
* Deoxynucleoside triphosphates (dNTPs) • Buffer
How much DNA do commercial PCR kits require?
Most commercial kits require 500-2500 pg of extracted DNA. Typically 1000 pg (1 ng) of total DNA in the PCR.
1 copy of the human genome is how many pg?
3
Before amplification what needs to happen to DNA?
It needs to be extracted and quantified
whats it called when less DNA is used?
low template DNA (LTDNA) or touch DNA
what are primers?
short synthetic pieces of DNA that anneal to the specific region of DNA
what do primers define?
the region of DNA to be analysed.
what are the requirements for primers?
- Complementary to conserved regions of DNA (human specific), so will amplify DNA
from all human populations. - Have similar melting temperature (Tm) of primers 40-60% GC content
- ~18-30 bases long
- Primer sequence should NOT contain self-complementary sequences, or sequence similarity between primers – produces secondary structures.
whats important when using primers in a multiplex?
length of the alleles
primers usually have what attached to them?
fluorescent dye
primers should not have what?
complementary sequence
what happens if there are complementary sequences between primers?
secondary structures
known as primer dimers are formed
self complementary sequence secondary structures lead to?
hair- pin structures forming
why should we avoid the formation of primer dimers and hair pin structures?
relatively small in size they are preferentially amplified over the target DNA.
primers are annealing to each other they are not annealing to the______ _____ and so reduced the _____ of the PCR.
target DNA
efficiency
How do we determine the annealing temperature of the oligonucleotide primers?
using calculations that take into consideration the sequence of the primer, the length and the salt concentration in the reaction
How can the optimum annealing temp be checked?
experimentally, too high the primers will fail to anneal successfully, too low you will get non- complementary hybridization.
what are PCR primers labelled with?
labelled with a fluorescent dye
why are PCR labelled with a fluorescent dye?
so that when PCR products are separated during electrophoresis the amplicons can be visualised
what happens in a multiplex reaction?
multiple target sites are amplified using several pairs of primers
amplicons produced by a multiplex reaction need to be separated by what?
size and fluorescent dye label
what does DNA polymerase do?
catalyses the attachment of deoxynucleoside triphosphates (dNTPs) to the single stranded template DNA
what polymerase has been used most for PCR?
Taq polymerase
polymerase enzymes need be what so they can function at the high temp of PCR?
thermally stable
polymerase can work ________ at room temperature.
non-specifically
whats a hot start reaction example?
- set up on ice
- manually separated from template
- primers modified so they do not work to reduce non specific activity
is Taq the only polymerase that can be used in PCR?
no
what are the main 4 properties of polymerase?
- Persistence/thermostability
- processivity
- fidelity
what does persistent refer to?
the stability of the enzyme at high temperatures
whats an issue with tax polymerase?
it’s activity can decrease with prolonged exposure to temperatures above 90 °C.
How do we overcome the Taq issues?
isolation of enzymes from hyperthermophilic organisms have been used.
is pfu polymerase more or less stable than taq polymerase at 95 degrees?
pfu 20 times more stable
persistence is the same as?
thermostability
what is Processivity ?
can be defined as the rate of the polymerase activity before the polymerase dissociates from the DNA.
Taq polymerase has a processivity of what?
processivity of 50-60 nucleotides (nt) per second at 72 °C.
Many other DNA polymerases have less processivity than Taq but have higher ____.
fidelity
what affects processivity?
salt concentration in the buffer and the DNA sequence.
what can Fidelity be defined as?
the accuracy of the complementary strand being formed.
It is important that the polymerase attaches the correct complementary nucleotide to the _____.
3ʹ terminus.
what is proof reading ?
Some polymerases possess a 3ʹ→5ʹ exonuclease activity known as proofreading, and can excise incorrect nucleotides and replace with the correct base.
High-fidelity polymerases would have what to give accurate amplification of the target DNA ?
low mis-incorporation rate and proofreading ability to give accurate amplification of the target DNA
when is low mis-incorporation rate and proofreading ability of polymerases important?
when trying to determine the sequence of a target
what are dNTPs
Building blocks used to extend the DNA.
what happens when dNTPs are added to a chain?
the two excess phosphates will be snipped off during polymerisation reaction.
When the new _____ is added to the 3ʹ end of the extending strand a ___ ______ and ______ ion is released.
nucleotide
pyrophosphate molecule
hydrogen
what does buffer do?
maintains pH and salt concentrations in the reaction
what type of salt does a buffer contain?
Contains monovalent salts such as potassium ions
what does magnesium chloride do?
stabilises the interaction between primer and template DNA,
allow the Taq polymerase to function
_____ _____ are essential cofactors for DNA polymerases.
Divalent ions
what is used to prevent PCR inhibition and increase yield?
Bovine serum albumin
what do commercial PCR kits come with?
Master Mix primer set size standard allelic ladder positive control
what is a positive control in PCR?
The positive control is a known DNA sample and the results from this can be checked to see if the PCR is working by comparing the genotype results.
what is a neg control in PCR?
A negative control is also used to check for contamination, here water or buffer are used instead of any template DNA.
what will you see with With samples that contain PCR inhibitors?
may see partial DNA profiles.
what can be the cause for the failure to amplify DNA?
due to reduced efficiency of the polymerase enzyme.
where do some PCR inhibitors come from?
co-extracted with the DNA from the sample itself eg: hematin from bloodstains.
where else can PCR inhibitors come from?
from extraction process e.g. chelating agents.
why is DNA neg charged?
Negatively charged due to the phosphate groups on the DNA backbone loosing hydrogen ions in
most buffer systems.
_________ is used to separate the PCR products by size
Electrophoresis
what are the two types of Electrophoresis?
- Slab-gel - Agarose and Polyacrylaminde (PAGE)
2. Capillary (refer to diagram )
In the main stages of a CE Electropherogram what happens?
- All peaks go through detection thresholds Analytical threshold (noise) Stochastic threshold
- Peak converted from time (min) to size (bp)
In CE Electropherogram whats the internal size standard?
DNA size (bp) given an allele call (number of repeats)
Genotype results are then analysed…. ?
manually by an experienced analyst.
Real time PCR can also indicate the presence of what?
PCR inhibitors and degraded DNA template.
Flourescence is monitored to indicate the amount of DNA, this can be done by ?
- The use of a fluorescent dye that binds to double stranded DNA.
- The use of a reporter probe that hybridizes to the specifically to the PCR product.
The amount of amplicons that accumulate during the exponential phase correlates to ?
the amount of starting material so can be used as a quantification technique.
what are some Biological artefacts of STR profiles?
- stutter
- Non-template nucleotide addition
- Microvariants – not within allelic ladder
- Mutations
- Null alleles
what I the biological artefact stutter?
slipped strand mis-pairing during PCR extension step. so extra peak, usually one repeat unit shorter. Than true allele peak.
How do you remove the peak caused by a stutter?
Use stutter filters (~15%) to remove this peak from reverse stutter/forward slippage.
what happens ta a Non-template nucleotide addition?
Polymerase adds an extra nucleotide to the 3ʹ-end (adenylation). Incomplete
adenylation can lead to split peaks typically caused by over amplification.
what happens with Microvariants – not within allelic ladder?
- Sequence variation compared to commonly observed alleles (nucleotide change) or an insertion/deletion (off-ladder (OL) alleles).
- False Tri-allelic patterns.
whats happens with mutations?
- Mutations can occur at the STR loci, STR have estimated mutation rates – germ-line mutations.
- Somatic mutations can occur (test different tissues).
what do Null alleles do?
Failure of amplification due to primer hybridisation problems.
what other type of amplification can be done?
whole genome amplification
Whole genome amplification can be done before or after our?
before
what is Whole genome amplification beneficial for?
low template DNA samples.
what does nested PCR do?
enrich the template by using two primer sets per target
nested PCR issue
this brings along problems with contamination.
How is RNA analysed?
- make DNA copy of the RNA using reverse transcription
- complementary DNA can then be amplified using PCR.
Analysis of RNA is useful in forensic ____ ___ ________ due the expression of certain genes in different tissue types.
body fluid identification
This profiling of RNA is commonly referred to as what?
everse transcriptase polymerase chain reaction (RT-PCR) using a two-step process.
