Multiplex PCR of STRs

Question 1

what are STRs

Answer 1

Short tandem repeats

Also called microsatellites

Variable, repeated DNA

Analysis on PCR

Question 2

what is PCR

Answer 2

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

Question 3

what year and who by was PCR invented

Answer 3

1985 by Kary Mullis

Question 4

what are the 4 steps of PCR

Answer 4

denaturation

annealing

extension

exponential amplification

Question 5

what is denaturation in PCR

Answer 5

temp increased to separate DNA strands

Question 6

what is annealing in PCR

Answer 6

temp is decreased to allow primers to base pair to complementary DNA template

Question 7

what is extension in PCR

Answer 7

polymerase extends primer to form nascent DNA strand

Question 8

what is exponential amplification

Answer 8

process is repeated, and the region of interest is amplified exponentially

Question 9

what are the 6 PCR components

Answer 9

Template DNA

Primers

Polymerase

Deoxynucleotide triphosphates (dNTPs) dATP, dTTP, dCTP, dGTP

Buffer – for optimum activity and stability of Taq

Ions

Question 10

DNA polymerase

Answer 10

Synthesises chain of nucleic acids

Utilised in PCR

Taq Polymerase

For PCR polymerase need to be thermostable

Question 11

What is Taq Polymerase named after

Answer 11

bacteria Thermus Aquaticus from hot springs

Question 12

DNA is anti-parallel

Answer 12

2 strands run parallel to each other but with opposite alignments

Provides a copying mechanism for the genetic material

Question 13

DNA replication

Answer 13

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

Question 14

stage 1 of DNA replication

Answer 14

helicases unwind the parental double helix

Question 15

stage 2 of DNA replication

Answer 15

single strand binding proteins stables the unwound parental DNA

Question 16

stage 3 of DNA replication

Answer 16

the leading strand is synthesised continuously in the 5’ to 3’ direction by DNA polymerase

Question 17

stage 4 of DNA replication

Answer 17

the lagging strand is synthesised discontinuously. primase synthesises a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment

Question 18

stage 5 of DNA replication

Answer 18

after the RNA primer is replaced by DNA, DNA ligase joins the Okazaki fragment to the growing strand

Question 19

primers

Answer 19

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

Question 20

what is a standard PCR cycle

Answer 20

28 cycles

Question 21

what is a thermocycler

Answer 21

can rapidly heat and cool samples to precise temp

Question 22

master mix

Answer 22

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

Question 23

input DNA

Answer 23

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)

Question 24

multiplex PCR

Answer 24

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

Question 25

forensic STR multiplexes

Answer 25

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

Question 26

size separation

Answer 26

Electrophoresis – gel or capillary If amplicons overlap in size you won’t be able to distinguish them

Question 27

what is an amplicon

Answer 27

a bit of DNA that's been amplified

Question 28

STR size

Answer 28

dependent on the number of repeat units

Question 29

what does amplicon size depend on

Answer 29

where the primers are placed - how close to the STR

Question 30

how can you make amplicons longer

Answer 30

place primers further from STR

Question 31

amplicon size

Answer 31

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

Question 32

how are amplicons the same size destinguished

Answer 32

by dye

Question 33

history of STR multiplexes

Answer 33

Quad (1994, FSS) SGM (1995, 6 STRs) SGM+ (2000, 10 STRs) DNA17 (2014, European Standard Set) International co-operation

Question 34

commercially available forensic multiplexes

Answer 34

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

Question 35

DNA 17

Answer 35

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

Question 36

Forensic Multiplex validation

Answer 36

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

Question 37

PCR contamination

Answer 37

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

Question 38

RFLP vs PCR

Answer 38

see powerpoint