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Bodies, tissues and fluids > Multiplex PCR of STR's > Flashcards

Multiplex PCR of STR's Flashcards

1
Q

STRs

A
  • Short tandem repeats
  • Also called microsatellites
  • Variable, repeated DNA
  • Analysis on PCR
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2
Q

PCR

A
  • 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
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3
Q

PCR process

A

denaturation
annealing
extension
exponential amplification

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4
Q

denaturation

A

temp is increased to separate DNA strands

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5
Q

annealing

A

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

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6
Q

extension

A

polymerase extends primer to form nascent DNA strand

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7
Q

exponential amplification

A

process is repeated and the region interest is amplified exponentially

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8
Q

PCR components

A
  • Template DNA
  • Primers
  • Polymerase
  • Deoxynucleotide tripohosphates (dNTPs) dATP, dTTP, dCTP, dGTP
  • Buffer- for optimum activity and stability of taq
  • Ions
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9
Q

DNA polymerase

A
  • Synthesises chains of nucleic acids
  • Utilised in PCR
  • Taq polymerase- bacteria from hot springs
  • For PCR polymerase needs to be thermo stable
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10
Q

DNA replication

A
  • 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

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11
Q

stage 1 of DNA replication

A

helices unwind the parental double helix

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12
Q

stage 2 of dna replication

A

single-strand binding proteins stabilise the unwound parental DNA

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13
Q

stage 3 of dna replication

A

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

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14
Q

stage 4 of dna replication

A

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

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15
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

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16
Q

primers

A
  • 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
17
Q

thermocycler

A

AB 9700. Can rapidly heat and cool samples to a precise temperature

18
Q

master mix

A
  • 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
19
Q

input DNA

A
  • 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)
20
Q

multiplex DNA

A
  • Amplify many targets in one reaction
  • > 1 set of primers
  • Save time and reagents
  • Various commercial forensic STR multiplex kits available
21
Q

multiplex development

A
  • 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
22
Q

forensics STR multiplexes

A
  • 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
23
Q

size separation

A
  • Electrophoresis- gel or capillary

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

24
Q

what is an amplicon

A

the bit of DNA that has been amplified

25
Q

amplicon size

A
  • 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
26
Q

what does amplicon size depend on

A

where the primers are placed how close to the STR

27
Q

how do you make amplicon longer

A

placing primers closer to STR

28
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 fluorescent dyes on primer to allow combination of more STRs
29
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
30
Q

forensic multiplex validation

A
  • 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
31
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
32
Q

RFLP vs PCR methods

A

look on powerpoint

Bodies, tissues and fluids (15 decks)

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