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Bodies, Tissues and fluids > Multiplex PCR of STRs > Flashcards

Multiplex PCR of STRs Flashcards

1
Q

what are STRs

A

Short tandem repeats

Also called microsatellites

Variable, repeated DNA

Analysis on PCR

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2
Q

what is PCR

A

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

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3
Q

what year and who by was PCR invented

A

1985 by Kary Mullis

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4
Q

what are the 4 steps of PCR

A

denaturation

annealing

extension

exponential amplification

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5
Q

what is denaturation in PCR

A

temp increased to separate DNA strands

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6
Q

what is annealing in PCR

A

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

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7
Q

what is extension in PCR

A

polymerase extends primer to form nascent DNA strand

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8
Q

what is exponential amplification

A

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

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9
Q

what are the 6 PCR components

A

Template DNA

Primers

Polymerase

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

Buffer – for optimum activity and stability of Taq

Ions

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10
Q

DNA polymerase

A

Synthesises chain of nucleic acids

Utilised in PCR

Taq Polymerase

For PCR polymerase need to be thermostable

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11
Q

What is Taq Polymerase named after

A

bacteria Thermus Aquaticus from hot springs

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12
Q

DNA is anti-parallel

A

2 strands run parallel to each other but with opposite alignments

Provides a copying mechanism for the genetic material

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13
Q

DNA replication

A

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

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14
Q

stage 1 of DNA replication

A

helicases unwind the parental double helix

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15
Q

stage 2 of DNA replication

A

single strand binding proteins stables the unwound parental DNA

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16
Q

stage 3 of DNA replication

A

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

17
Q

stage 4 of DNA replication

A

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

18
Q

stage 5 of DNA replication

A

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

19
Q

primers

A

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

20
Q

what is a standard PCR cycle

A

28 cycles

21
Q

what is a thermocycler

A

can rapidly heat and cool samples to precise temp

22
Q

master mix

A

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

23
Q

input DNA

A

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)

24
Q

multiplex PCR

A

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

25
Q

forensic STR multiplexes

A

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

26
Q

size separation

A

Electrophoresis – gel or capillary

If amplicons overlap in size you won’t be able to distinguish them

27
Q

what is an amplicon

A

a bit of DNA that’s been amplified

28
Q

STR size

A

dependent on the number of repeat units

29
Q

what does amplicon size depend on

A

where the primers are placed - how close to the STR

30
Q

how can you make amplicons longer

A

place primers further from STR

31
Q

amplicon size

A

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

32
Q

how are amplicons the same size destinguished

A

by dye

33
Q

history of STR multiplexes

A

Quad (1994, FSS)

SGM (1995, 6 STRs)

SGM+ (2000, 10 STRs)

DNA17 (2014, European Standard Set)

International co-operation

34
Q

commercially available forensic multiplexes

A

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

35
Q

DNA 17

A

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

36
Q

Forensic Multiplex validation

A

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

37
Q

PCR contamination

A

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

38
Q

RFLP vs PCR

A

see powerpoint

Bodies, Tissues and fluids (16 decks)

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