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2nd year sem 1: Forensic Genetics > Lecture Two: Polymerase chain reaction and forensic DNA profiling (finished) > Flashcards

Lecture Two: Polymerase chain reaction and forensic DNA profiling (finished) Flashcards

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1
Q

what was PCR first used for?

A

to identify skeletal remains using HLA DQα locus

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2
Q

At first to use PCR on VNTRs the sample had to be what?

A

fresh

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3
Q

By the 1990s PCR was used for what?

A

was used to amplify STRs

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4
Q

whats another word for

Human minisatellites?

A

variable number tandem repeats

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5
Q

How many bp’s do variable number tandem repeats have?

A

6-100bp

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6
Q

Amplified Fragment length Polymorphisms tandem repeats have an amplicon size smaller than what?

A

smaller than 1 kb to allow successful amplification

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7
Q

Human microsatellites are also known as what?

A

short tandem repeats

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8
Q

what stem typical core repeat for a STR?

A

2-6bp

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9
Q

whats the amplicon size of a STR?

A

Amplicon size 100-500 bp.

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10
Q

whats a Dinucleotide repeat?

A

same two repeat, example : (CA)(CA)(CA)

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11
Q

whats a simple repeat?

A

Tandem repeats with identical repeat units.

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12
Q

what do Non-consensus Alleles (microvariants) contain?

A

Contain incomplete repeats by one or more nucleotides

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13
Q

whats a

Compound Repeat?

A

Consist of more than one type of simple repeat.

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14
Q

what does a Complex Repeat contain?

A

Contain several clusters of different tandem repeats with intervening sequences.

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15
Q

whats a simple definition for PCR?

A

Repeated copying of a selected region of a DNA molecule.

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16
Q

where is PCR done?

A

in vitro

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17
Q

who won the noble prize for PCR?

A

Kary Mullis in 1985

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18
Q

what are the 3 main steps of PCR?

A

Denaturation
Annealing
Extension

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19
Q

what happens at denaturation?

A

Denaturation at high

temperature (92°C-95°C)

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20
Q

what happens at annealing?

A 
Annealing at a cooler
annealing temperature (50°C-65°C)(~5°C below primers’ Tm; optimised experimentally)
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21
Q

what happens at extension ?

A

Extension at a temperature between the annealing and denaturing temperatures (usually at 72°C)

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22
Q

whats one cycle of PCR?

A

denature- heat to separate strands
annealing - hybridisation of primers
extension - DNA synthesis from primers

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23
Q

whats the average cycle length in PCR?

A

25-35 cycles

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24
Q

at what cycle is the correct size double stranded target created?

A

cycle 3

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25
Q

what is adenylation (A tailing)?

A

adding of a non-template nucleotide to the 3ʹ end.

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26
Q

what does incomplete adenylation show as?

A

this will appear as a split peak on the

electropherogram (epg) due to the different size PCR products.

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27
Q

what amplicon is favoured for adenylation?

A

+A amplicon

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28
Q

In adenylation how do you go from -A amplicon to +A amlicon?

A

by a final incubation step.

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29
Q

what are the three phases of a PCR amplification curve?

A

Exponential
Linear
Plateau

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30
Q

Till when does the exponential phase continue?

A

The exponential phase will continue until one or more of the components of the reaction become limited.

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31
Q

what does the linear phase in the PCR amplification curve show?

A

The linear phase is an indication that the amplification efficiency is decreasing.

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32
Q

what happens to the plateau phase in the PCR amplification curve?

A

The plateau phase is where no new amplicons are accumulating due to the exhaustion of reagents.

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33
Q

what are the 5 components of PCR?

A
  • Template DNA
  • Oligonucleotide primers
  • DNA polymerase
  • Deoxynucleoside triphosphates (dNTPs) • Buffer
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34
Q

what does a PCR commercial master mix contain?

A
  • DNA polymerase

* Deoxynucleoside triphosphates (dNTPs) • Buffer

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35
Q

How much DNA do commercial PCR kits require?

A

Most commercial kits require 500-2500 pg of extracted DNA. Typically 1000 pg (1 ng) of total DNA in the PCR.

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36
Q

1 copy of the human genome is how many pg?

A

3

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37
Q

Before amplification what needs to happen to DNA?

A

It needs to be extracted and quantified

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38
Q

whats it called when less DNA is used?

A

low template DNA (LTDNA) or touch DNA

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39
Q

what are primers?

A

short synthetic pieces of DNA that anneal to the specific region of DNA

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40
Q

what do primers define?

A

the region of DNA to be analysed.

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41
Q

what are the requirements for primers?

A
  1. Complementary to conserved regions of DNA (human specific), so will amplify DNA
    from all human populations.
  2. Have similar melting temperature (Tm) of primers 40-60% GC content
  3. ~18-30 bases long
  4. Primer sequence should NOT contain self-complementary sequences, or sequence similarity between primers – produces secondary structures.
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42
Q

whats important when using primers in a multiplex?

A

length of the alleles

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43
Q

primers usually have what attached to them?

A

fluorescent dye

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44
Q

primers should not have what?

A

complementary sequence

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45
Q

what happens if there are complementary sequences between primers?

A

secondary structures

known as primer dimers are formed

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46
Q

self complementary sequence secondary structures lead to?

A

hair- pin structures forming

47
Q

why should we avoid the formation of primer dimers and hair pin structures?

A

relatively small in size they are preferentially amplified over the target DNA.

48
Q

primers are annealing to each other they are not annealing to the______ _____ and so reduced the _____ of the PCR.

A

target DNA

efficiency

49
Q

How do we determine the annealing temperature of the oligonucleotide primers?

A

using calculations that take into consideration the sequence of the primer, the length and the salt concentration in the reaction

50
Q

How can the optimum annealing temp be checked?

A

experimentally, too high the primers will fail to anneal successfully, too low you will get non- complementary hybridization.

51
Q

what are PCR primers labelled with?

A

labelled with a fluorescent dye

52
Q

why are PCR labelled with a fluorescent dye?

A

so that when PCR products are separated during electrophoresis the amplicons can be visualised

53
Q

what happens in a multiplex reaction?

A

multiple target sites are amplified using several pairs of primers

54
Q

amplicons produced by a multiplex reaction need to be separated by what?

A

size and fluorescent dye label

55
Q

what does DNA polymerase do?

A

catalyses the attachment of deoxynucleoside triphosphates (dNTPs) to the single stranded template DNA

56
Q

what polymerase has been used most for PCR?

A

Taq polymerase

57
Q

polymerase enzymes need be what so they can function at the high temp of PCR?

A

thermally stable

58
Q

polymerase can work ________ at room temperature.

A

non-specifically

59
Q

whats a hot start reaction example?

A
  • set up on ice
  • manually separated from template
  • primers modified so they do not work to reduce non specific activity
60
Q

is Taq the only polymerase that can be used in PCR?

A

no

61
Q

what are the main 4 properties of polymerase?

A
  1. Persistence/thermostability
  2. processivity
  3. fidelity
62
Q

what does persistent refer to?

A

the stability of the enzyme at high temperatures

63
Q

whats an issue with tax polymerase?

A

it’s activity can decrease with prolonged exposure to temperatures above 90 °C.

64
Q

How do we overcome the Taq issues?

A

isolation of enzymes from hyperthermophilic organisms have been used.

65
Q

is pfu polymerase more or less stable than taq polymerase at 95 degrees?

A

pfu 20 times more stable

66
Q

persistence is the same as?

A

thermostability

67
Q

what is Processivity ?

A

can be defined as the rate of the polymerase activity before the polymerase dissociates from the DNA.

68
Q

Taq polymerase has a processivity of what?

A

processivity of 50-60 nucleotides (nt) per second at 72 °C.

69
Q

Many other DNA polymerases have less processivity than Taq but have higher ____.

A

fidelity

70
Q

what affects processivity?

A

salt concentration in the buffer and the DNA sequence.

71
Q

what can Fidelity be defined as?

A

the accuracy of the complementary strand being formed.

72
Q

It is important that the polymerase attaches the correct complementary nucleotide to the _____.

A

3ʹ terminus.

73
Q

what is proof reading ?

A

Some polymerases possess a 3ʹ→5ʹ exonuclease activity known as proofreading, and can excise incorrect nucleotides and replace with the correct base.

74
Q

High-fidelity polymerases would have what to give accurate amplification of the target DNA ?

A

low mis-incorporation rate and proofreading ability to give accurate amplification of the target DNA

75
Q

when is low mis-incorporation rate and proofreading ability of polymerases important?

A

when trying to determine the sequence of a target

76
Q

what are dNTPs

A

Building blocks used to extend the DNA.

77
Q

what happens when dNTPs are added to a chain?

A

the two excess phosphates will be snipped off during polymerisation reaction.

78
Q

When the new _____ is added to the 3ʹ end of the extending strand a ___ ______ and ______ ion is released.

A

nucleotide
pyrophosphate molecule
hydrogen

79
Q

what does buffer do?

A

maintains pH and salt concentrations in the reaction

80
Q

what type of salt does a buffer contain?

A

Contains monovalent salts such as potassium ions

81
Q

what does magnesium chloride do?

A

stabilises the interaction between primer and template DNA,

allow the Taq polymerase to function

82
Q

_____ _____ are essential cofactors for DNA polymerases.

A

Divalent ions

83
Q

what is used to prevent PCR inhibition and increase yield?

A

Bovine serum albumin

84
Q

what do commercial PCR kits come with?

A 
Master Mix
primer set
 size standard
 allelic ladder
 positive control
85
Q

what is a positive control in PCR?

A

The positive control is a known DNA sample and the results from this can be checked to see if the PCR is working by comparing the genotype results.

86
Q

what is a neg control in PCR?

A

A negative control is also used to check for contamination, here water or buffer are used instead of any template DNA.

87
Q

what will you see with With samples that contain PCR inhibitors?

A

may see partial DNA profiles.

88
Q

what can be the cause for the failure to amplify DNA?

A

due to reduced efficiency of the polymerase enzyme.

89
Q

where do some PCR inhibitors come from?

A

co-extracted with the DNA from the sample itself eg: hematin from bloodstains.

90
Q

where else can PCR inhibitors come from?

A

from extraction process e.g. chelating agents.

91
Q

why is DNA neg charged?

A

Negatively charged due to the phosphate groups on the DNA backbone loosing hydrogen ions in
most buffer systems.

92
Q

_________ is used to separate the PCR products by size

A

Electrophoresis

93
Q

what are the two types of Electrophoresis?

A
  1. Slab-gel - Agarose and Polyacrylaminde (PAGE)

2. Capillary (refer to diagram )

94
Q

In the main stages of a CE Electropherogram what happens?

A
  • All peaks go through detection thresholds Analytical threshold (noise) Stochastic threshold
  • Peak converted from time (min) to size (bp)
95
Q

In CE Electropherogram whats the internal size standard?

A

DNA size (bp) given an allele call (number of repeats)

96
Q

Genotype results are then analysed…. ?

A

manually by an experienced analyst.

97
Q

Real time PCR can also indicate the presence of what?

A

PCR inhibitors and degraded DNA template.

98
Q

Flourescence is monitored to indicate the amount of DNA, this can be done by ?

A
  1. The use of a fluorescent dye that binds to double stranded DNA.
  2. The use of a reporter probe that hybridizes to the specifically to the PCR product.
99
Q

The amount of amplicons that accumulate during the exponential phase correlates to ?

A

the amount of starting material so can be used as a quantification technique.

100
Q

what are some Biological artefacts of STR profiles?

A
  • stutter
  • Non-template nucleotide addition
  • Microvariants – not within allelic ladder
  • Mutations
  • Null alleles
101
Q

what I the biological artefact stutter?

A

slipped strand mis-pairing during PCR extension step. so extra peak, usually one repeat unit shorter. Than true allele peak.

102
Q

How do you remove the peak caused by a stutter?

A

Use stutter filters (~15%) to remove this peak from reverse stutter/forward slippage.

103
Q

what happens ta a Non-template nucleotide addition?

A

Polymerase adds an extra nucleotide to the 3ʹ-end (adenylation). Incomplete
adenylation can lead to split peaks typically caused by over amplification.

104
Q

what happens with Microvariants – not within allelic ladder?

A
  • Sequence variation compared to commonly observed alleles (nucleotide change) or an insertion/deletion (off-ladder (OL) alleles).
  • False Tri-allelic patterns.
105
Q

whats happens with mutations?

A
  • Mutations can occur at the STR loci, STR have estimated mutation rates – germ-line mutations.
  • Somatic mutations can occur (test different tissues).
106
Q

what do Null alleles do?

A

Failure of amplification due to primer hybridisation problems.

107
Q

what other type of amplification can be done?

A

whole genome amplification

108
Q

Whole genome amplification can be done before or after our?

A

before

109
Q

what is Whole genome amplification beneficial for?

A

low template DNA samples.

110
Q

what does nested PCR do?

A

enrich the template by using two primer sets per target

111
Q

nested PCR issue

A

this brings along problems with contamination.

112
Q

How is RNA analysed?

A
  • make DNA copy of the RNA using reverse transcription

- complementary DNA can then be amplified using PCR.

113
Q

Analysis of RNA is useful in forensic ____ ___ ________ due the expression of certain genes in different tissue types.

A

body fluid identification

114
Q

This profiling of RNA is commonly referred to as what?

A

everse transcriptase polymerase chain reaction (RT-PCR) using a two-step process.

2nd year sem 1: Forensic Genetics (8 decks)

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