Multiplex PCR of STR's Flashcards
STRs
- Short tandem repeats
- Also called microsatellites
- Variable, repeated DNA
- Analysis on PCR
PCR
- Polymerase chain reaction
- Invented in 1985 by kary mullis
- Revolutionised molecular biology
- Millions of copies of a DNA sequence can be made in a few hours
- Enzyme based- DNA polymerase
- Mimics DNA replication in the body
PCR process
denaturation
annealing
extension
exponential amplification
denaturation
temp is increased to separate DNA strands
annealing
temp is decreased to allow primers to base pair to complementary DNA template
extension
polymerase extends primer to form nascent DNA strand
exponential amplification
process is repeated and the region interest is amplified exponentially
PCR components
- Template DNA
- Primers
- Polymerase
- Deoxynucleotide tripohosphates (dNTPs) dATP, dTTP, dCTP, dGTP
- Buffer- for optimum activity and stability of taq
- Ions
DNA polymerase
- Synthesises chains of nucleic acids
- Utilised in PCR
- Taq polymerase- bacteria from hot springs
- For PCR polymerase needs to be thermo stable
DNA replication
- DNA is antiparallel
- DNA polymerases 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’->3’
- Mechanism also allows for repair of DNA strands
- Repair of mismatched bases during replication
- repair of damage to DNA form physical and chemical attack
for more detailed go to power point
stage 1 of DNA replication
helices unwind the parental double helix
stage 2 of dna replication
single-strand binding proteins stabilise the unwound parental DNA
stage 3 of dna replication
the leading strand is synthesised continuously in the 5’->3’ direction by DNA polymerase
stage 4 of dna replication
the lagging strand is synthesised discontinuously primate synthesises a short RNA primer, which is extended by DNA polymerase to form 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 (approx. 10 bp) that flank the sequence to be copied and act as a target for the DNA polymerase
- Design primers specific for your template, chemically synthesised
- DNA polymerase adds nucleotides to the primer to synthesise DNA (amplicon)
- Directionality of DNA
- Forward & reverse primers
- Primers always come in pairs so you can make copies of both of the strands
thermocycler
AB 9700. Can rapidly heat and cool samples to a precise temperature
master mix
- Pre mixed, ready to use solution containing PCR components
- Ensures uniformity of conditions for samples
- Total reaction volume usually 25 microlitres or 50 microlitres
- Template DNA= 0.5µl (not included in Master Mix)
- Add 24.5µl of Master Mix to each tube
- Then add template:
- Sample= extracted DNA from sample
- Positive control= clean extracted DNA (supplied)
- Negative control= water
input DNA
- Most PCR systems require 0,5ng- 2.5ng of extracted template DNA
- Equivalent to approx. 166-833 copies of the genome
- If you’ve quantified the DNA you can calculate what volume will give you the optimum input (make up difference with water)
multiplex DNA
- Amplify many targets in one reaction
- > 1 set of primers
- Save time and reagents
- Various commercial forensic STR multiplex kits available
multiplex development
- Creating a multiplex can be as simple as combining two sets of primers
- May involve tedious optimisation experiments
- Optimisation mainly related to primers
- Primer pairs need to be compatible
- Annealing and melting temperatures
- Avoid excessive regions of complementarity to avoid primer-dimers
- Primer length
- Primer- BLAST
forensics STR multiplexes
- Co amplify STRs of 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
size separation
- Electrophoresis- gel or capillary
- If the amplicons overlap in size you won’t be able to distinguish them
what is an amplicon
the bit of DNA that has been amplified
amplicon size
- STR itself has a size dependent on the number or repeat units
- when designing a forensic STR multiplex u want a range of amplicon sizes (100-450bp)
- limits the number of loci
- by adding fluorescent dyes to the primers you can increase the number of loci
- several amplicons may have the same size but will be distinguished by dye
what does amplicon size depend on
where the primers are placed how close to the STR
how do you make amplicon longer
placing primers closer to STR
commercially available forensic multiplexes
- 10-24 loci
- SGM+, DNA-17, CODIS, Powerplex 21, Globalfiler
- Sold as kits- easy to prepare reaction
- International standardisation
- Use fluorescent dyes on primer 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 lab it should be fully validated
- Establishing, though 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 methods
look on powerpoint
