Multiplex PCR of STRs Flashcards
what are STRs
Short tandem repeats
Also called microsatellites
Variable, repeated DNA
Analysis on PCR
what is PCR
Polymerase chain reaction
Millions of copies of a DNA sequence can be made in a few hours
Enzyme based – DNA polymerase
Mimics DNA replication in the body
Many forensic samples have such low levels of DNA or degraded DNA that would not be able to analyse without amplification via PCR
what year and who by was PCR invented
1985 by Kary Mullis
what are the 4 steps of PCR
denaturation
annealing
extension
exponential amplification
what is denaturation in PCR
temp increased to separate DNA strands
what is annealing in PCR
temp is decreased to allow primers to base pair to complementary DNA template
what is extension in PCR
polymerase extends primer to form nascent DNA strand
what is exponential amplification
process is repeated, and the region of interest is amplified exponentially
what are the 6 PCR components
Template DNA
Primers
Polymerase
Deoxynucleotide triphosphates (dNTPs) dATP, dTTP, dCTP, dGTP
Buffer – for optimum activity and stability of Taq
Ions
DNA polymerase
Synthesises chain of nucleic acids
Utilised in PCR
Taq Polymerase
For PCR polymerase need to be thermostable
What is Taq Polymerase named after
bacteria Thermus Aquaticus from hot springs
DNA is anti-parallel
2 strands run parallel to each other but with opposite alignments
Provides a copying mechanism for the genetic material
DNA replication
DNA polymerase add nucleotides only to the free 3’ end of a DNA molecule, never to the 5’ end
A new DNA strand can only be replicated 5’ to 3’
Mechanism also allows for repair of DNA strands
Repairs of mismatched bases during replication
Repair of damage to DNA from physical and chemical attack
stage 1 of DNA replication
helicases unwind the parental double helix
stage 2 of DNA replication
single strand binding proteins stables the unwound parental DNA
stage 3 of DNA replication
the leading strand is synthesised continuously in the 5’ to 3’ direction by DNA polymerase
stage 4 of DNA replication
the lagging strand is synthesised discontinuously. primase synthesises a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment
stage 5 of DNA replication
after the RNA primer is replaced by DNA, DNA ligase joins the Okazaki fragment to the growing strand
primers
Short DNA sequences (10bp) that flank the sequence to be copied and act as a target for DNA polymerase
Design primers specific for your template, chemically synthesised
DNA polymerase adds nucleotides to the primer to synthesis DNA (amplicon = PCR product)
Directionality of DNA
Forwards and reverse primer
what is a standard PCR cycle
28 cycles
what is a thermocycler
can rapidly heat and cool samples to precise temp
master mix
Pre-mixed, ready-to-use solution containing PCR components
Ensures uniformity of conditions for samples
Total reaction volume usually 25ul or 50ul
No DNA in the master mix
input DNA
Most PCR systems require 0.5ng-2.5ng of extracted template DNA
Equivalent to 166-833 copies of genome
If you have quantified, you DNA you can calculate what volume will give you the optimum input (make up difference with water)
multiplex PCR
Amplify many targets in one reaction
> 1 set of primers
Save time and reagents
Various commercial forensic STR multiplex kits available
Annealing and melting temperatures
Avoid excessive regions of complementarity to avoid primer-dimers
Primer length
Primer-BLAST
forensic STR multiplexes
Co-amplify STRs interest to the forensic community to produce a DNA profile
Need good size separation – amplicon length
No non-specific products that might interfere with interpretation of DNA profile
size separation
Electrophoresis – gel or capillary
If amplicons overlap in size you won’t be able to distinguish them
what is an amplicon
a bit of DNA that’s been amplified
STR size
dependent on the number of repeat units
what does amplicon size depend on
where the primers are placed - how close to the STR
how can you make amplicons longer
place primers further from STR
amplicon size
When designing a forensic STR multiplex, want a range of amplicon sizes
Limits the number of loci
But by adding fluorescent dyes to the primers you can increase the number of loci
how are amplicons the same size destinguished
by dye
history of STR multiplexes
Quad (1994, FSS)
SGM (1995, 6 STRs)
SGM+ (2000, 10 STRs)
DNA17 (2014, European Standard Set)
International co-operation
commercially available forensic multiplexes
10-24 loci
SGM+, DNA-17, CODIS, PowerPlex 21, Globalfiler
Sold as kits – easy to prepare reaction
International standardisation
Use of fluorescent dyes on primers to allow combination of more STRs
DNA 17
From July 2014 using the DNA17 system
Compatible with previous SGM+ system for NDNAD
Development of European Standard Set (ESS) of loci for more cross-border collaboration
Increased sensitivity
Forensic Multiplex validation
Before introducing any new technique into a forensic laboratory, it should be fully validated
Establishing, through documented experimentation, that a scientific method or technique is fit for its intended purpose.
On a practical level how multiplex will fit into lab workflow and what impact it will have on results
Define the limitations of the technique
Reaction volume
Interpretation guidelines
PCR cycling
PCR contamination
The sensitivity of PCR makes contamination an issue
Keep pre & post-PCR samples separate
Use appropriate PPE
Set up reaction in laminar flow hood
Use aerosol resistant pipette tips & change for each new sample
RFLP vs PCR
see powerpoint
