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Study Notes 3rd Year > FRSC 3000 Lab DNA > Flashcards

FRSC 3000 Lab DNA Flashcards

1
Q

Advantages of PCR based methods

A

Very small amounts of DNA template may be used

Effective templates for amplification

Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR

Contaminant DNA will not amplify because human-specific primers are used

Commercial kits are now available for easy PCR reaction setup and amplification

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2
Q

Disadvantages of PCR based methods

A

The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA

Amplification may fail due to sequence mutations in the primer-binding region of the genomic DNA template – ‘null allele’

Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without the use of careful laboratory technique and validated protocols

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3
Q

AFLP

A

Amplified fragment length polymorphism

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4
Q

Advantages and disadvantages to D1S80 AFLP

A

Advantages:
Improved sensitivity compared to RFLP because it uses PCR.
Many alleles which facilitates mixed-sample analysis.
Discrete allele calling possible using allelic ladder, which also simplifies statistical interpretation.

Limitations:
Large allele range making it difficult to multiplex with other loci and giving rise to preferential amplification of smaller alleles.
Poor power of discrimination as a single locus (≈1 in 50).
Allele dropout seen with highly degraded DNA.
Gel separation and silver-stain detection not amenable to automation or high-throughput sample processing.

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5
Q

About DQA1 Reverse Blot tests

A

1st methods used allele specific probes to find sequence polymorphisms in a dot-blot format

Most common locus was HLA-DQA1 (codes for integral membrane protein associated with immune response)

Relies on hybridization of sample PCR products to test allele dots

Probe that is bound to the membrane is specific for one of the possible alleles

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6
Q

Limitations of reverse blot tests

A

Poor power of discrimination (≈1 in 1000) with only six loci developed each containing only a few alleles.

Mixture interpretation is difficult with a limited number of alleles per locus.

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7
Q

STR

A

Short tandem repeat

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8
Q

Causes of STRs

A

replication slippage
- occurs at a repetitive sequence when the new strand mis-pairs with the template strand

If occurs at the coding region it could cause diseases

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9
Q

Silver stained STRS detection and advantages and disadvantages

A

detection with CTT complex

Advantages:
Sensitive due to PCR.
Relatively rapid process (a day or two).
Works well with degraded DNA samples since shorter fragments of DNA can be analyzed (compared to D1S80).
A lower start-up cost compared to fluorescent STRs

Limitations:
Because only a single ‘color’ channel is available, multiplex amplification and detection is limited to 3 to 4 loci
Both strands of DNA are detected leading to double bands with some loci that can complicate interpretation

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10
Q

What is CCT complex

A

CSF1PO
TPOX
TH01

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11
Q

Fluorescent STRs

A

PCR primers anneal to unique sequences bracketing the variable STR repeat region

The overall PCR product is measured

Fluorescent dye primer, therefore when amplified, that dye will be incorporated into the area of interest

Then use capillary electrophoresis, and analyze the amplified gene

Ladder is included for reference sample

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12
Q

Advantages of fluorescent STRs

A

Sensitive due to PCR (single cell analysis has been demonstrated).

Relatively rapid process (can be completed in a few hours or at most a day or two).

Works fairly well with degraded samples since shorter fragments of DNA can be analyzed (compared to D1S80). miniSTRs have extended the capabilities for degraded sample analysis.

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13
Q

Disadvantages of Fluorescent STRs

A

Less discrimination power per locus compared to VNTRs due to a smaller number of alleles and less heterozygosity per locus.

The possibility of contamination from stray DNA is increased because of the PCR amplification process.

Expensive equipment required for detection

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14
Q

Compare RFLP and PCR

A

RFLP:
6-8 weeks

you need 50-500ng of DNA

Binning is required fir allele identification

Must be double stranded

PCR:
1-2 days

Need 0.1-1ng of DNA

Discrete alleles are obtained

DNA can be either single or double stranded

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15
Q

Non-DNA based technology (2)

A

Blood group testing

Forensic protein profiling

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16
Q

DNA based technology (4)

A

RFLP

PCR based (sequence)

PCR based (length)

Mitochondrial DNA sequencing

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17
Q

DNA technology with low power of discrimination and slow speed of analysis

A

mtDNA

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18
Q

DNA technology with low power of discrimination and fast speed of analysis

A

Polymarker D1S280 single STR

ABO blood groups

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19
Q

DNA technology with high power discrimination and fast speed of analysis

A

Multiplex STRs

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20
Q

DNA technology with high power discrimination and slow speed of analysis

A

RFLP multi-locus probes

RFLP single-locus probes

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21
Q

Process of RFLP based DNA testing

A

Cut DNA with restriction enzymes

Separate fragments differing in length by gel electrophoresis

Detect length-based differences (polymorphisms) in DNA fragments of interest with a radioactive probe

Strip membrane and re-probe as necessary

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22
Q

Southern Blot and RFLP

A

Individuals are tested and blood samples are taken

DNA is extracted form white blood cells

RE is added

Smear of genomic DNA is created

DNA is denatured

Weight is applied (southern transfer process)

Filter receives single stranded DNA replica of gel

Hybridization

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23
Q

Locus

A

Specific region of DNA

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24
Q

Alleles

A

Alternative forms of a locus (A or a)

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25
Q

Homozygous

A

If two alleles are identical by descent at a locus

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26
Q

heterozygous

A

If two alleles are different by descent at a locus

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27
Q

Genotype

A

the characterization of alleles at a locus (AA or Bb)

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28
Q

DNA profile

A

the genotypes obtained at multiple loci (AABb)

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29
Q

How many chromosomes does a human have

A

22 pairs of chromosomes plus the XX or XY

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30
Q

DNA structure

A

includes sugar backbone, phosphate groups and four nucleotide bases

Phosphodiester bonds between adjacent nucleotides

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31
Q

What causes DNA to denature

A

Elevated temperatures or chemical treatments by disrupting hydrogen bonds i.e. formamide/salts

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32
Q

2 types of DNA polymorphisms

A

Sequence

Length

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33
Q

To be useful DNA markers must:

A

Variable (polymorphic)

Reproducible detection

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34
Q

Who developed DNA profiling

A

Alec Jefferys 1985

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35
Q

VNTR

A

Variable number of tandem repeats

Analyzed using RFLPs

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36
Q

3 possible outcomes of evidence examination

A

Exclusion - No match

Non-exclusion - Match or inclusion

Inconclusive - no result

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37
Q

Defense case in the OJ Simpson trial

A

Attacked the way evidence was collected and preserved

The defense had brought forward the way in which the DNA was collected. They had stated that during DNA collection, it was contaminated, causing identification to be incorrect as there could have been a mix. They also challenged that the DNA was preserved improperly and that contamination must have occurred when handling the evidence improperly and had mixed up suspects.

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38
Q

The basic rules of evidence collection and preservation

A

Avoid contamination form the collector

Seperatly package evidence

Air dry “wet” samples prior to packaging in paper DO NOT use plastic

All samples must be properly labelled and sealed

Stains on unmovable surfaces must be swabbed

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39
Q

What are presumptive tests used for and what are some requirements

A

to indicate whether biological fluids are present on an item of evidence

Should be:
Simple
Inexpensive
Safe
Only use a small amount of material
non-destructive

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40
Q

3 things presumptive tests, test for

A

Blood - serological methdods to detect hemoglobin or luminol

Semen - Serological methods to detect acid phosphatase or direct observation

Saliva - Serological methods to detect amylase

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41
Q

Advantages of body fluid identification using mRNA testing

A

high sensitivity

High specificity

simultaneous DNA isolation without loss of material

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42
Q

What DNA transcript can be used as a positive RNA control

A

“housekeeping genes”

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43
Q

Steps to DNA extraction

A

Lyse cells and release DNA

Separate DNA from cellular material (digest bound proteins )

Isolate DNA so that it can be used for downstream

STR typing (final solution looks like a tube of water)

Store DNA at -20 oC or -80 oC to prevent nuclease activity!!

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44
Q

Goal of DNA extraction

A

maximize yield while minimizing contaminants/inhibitors from other cellular materials

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45
Q

Solution of Iron from RBC PCR inhibiton

A

Centrifugation

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46
Q

Solution of Ethanol PCR inhibitor

A

Heat elution with lid off

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47
Q

Solution to Minerals from bone PCR inhibitor

A

Decalcify prior to extraction

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48
Q

Solution to indigo dye, melanin, and protein PCR inhibitors

A

Dilution

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49
Q

What does increased hydrolytic cleavage of DNA due to high temperatures cause

A

nicks in the DNA tempate which can interfere with primer annealing or Taq polymerase

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50
Q

5 Extraction methods

A

Organic, Solid-phase (qiagen), Chelex, FTA paper, Differential extraction

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51
Q

Organic Extractions

A

cell lysate is treated with buffer-saturated phenol chloroform

DNA remains in aqueous phase while cellular proteins are denatures and precipitated

DNA is then treated with isopropanol or ethanol to precipitate DNA

Precipitated DNA is washed with ethanol then rehydrated with TE

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52
Q

Solid-phase DNA extraction

A

Higher sample throughput via automation of DNA extraction

DNA selectively binds to a substrate (silica resin or silica coated magnetic bead)

DNA is retained while proteins and cellular debris are washed away

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53
Q

Qiagen Extraction

A

Nucleic acids bind to a porous silicon-oxide (SiO- ) coated membrane in the presence of chaotropic salts (e.g., guanidine hydrochloride) and ethanol under slightly acidic conditions

Washing steps in the presence of chaotropic salt and ethanol keep proteins/cellular debris in solution so they can ‘flow through’ column, while DNA is bound to the column

Under low salt conditions the DNA is eluted (released) from the support into solutio

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54
Q

Promega Extraction

A

Uses magnetic beads (a silicon-oxide coated paramagnetic resin) to bind DNA

Adding resin to the lysate allows extraction to be performed in one tube; various solutions are used to wash the magnetic beads and a magnet holds DNA in place

The amount of resin used is the limiting factor to amount of DNA captured

At the final step, the sample is incubated at 65oC in an elution buffer (normally TE), which releases the DNA from the beads into solution

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55
Q

Differential extraction

A

Goal: separate epithelial cells from sperm cells in sexual assault cases to facilitate STR interpretation

Modified organic extraction that preferentially breaks open female epithelial cells (no DTT in 1st digestion)

Sperm cells then lysed with DTT (dithiothreitol) that breaks protein disulfide bridges of sperm cell walls

Not useful for azoospermic sperm (e.g., vasectomy; where there are no measurable levels of sperm)

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56
Q

What does too much DNA in quantitation cause

A

Off-scale peaks
Split peaks (+/-A)
Locus-to-locus imbalance

Therefore you Must dilute sample

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57
Q

What does too little DNA in quantitation cause

A

Heterozygote peak imbalance
Allele drop-out
Locus-to-locus imbalance

Therefore you, Must concentrate sample

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58
Q

Slot Blot method

A

most used method during late 90s

Primate specific prove binds to satellite sequence

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59
Q

What wavelength do DNA and RNA absorb UV light

A

maximum of 260nm

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60
Q

What is considered a poor limit of detection

A

~3.5ng/ul

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61
Q

Pico Green Assay

A

Detection is 50pg/ul-2ug/ul

Uses a standard curve to convert fluorescence signal into the amount of DNA present in unknown sample

Not human specific and only dsDNA specific

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62
Q

What is the difference between PCr and Quantitative PCR

A

In PCR the products are analyzed after the cycling is completed

In quantitative PCR (qPCR) the products are monitored as the PCR is occurring

Once per thermal cycle, fluorescence is measured and recorded as a normalized reporter signal (Rn)

We monitor the PCR during the exponential phase where first significant increase in amount of PCR product correlates to initial amount of target template

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63
Q

what is Ct

A

Cycle threshold which is defined as the number of cycles required for the fluorescent signal to exceed background levels

low Ct means a greater amount of nucleic acid in sample

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64
Q

Two main types of Quantitative PCR assays

A

fluorogenic 5’ nuclease

Inter-chelating dye SYBR green

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65
Q

5’ Nuclease Assay

A

TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites

The miner groove binder at the 3’ end of the probe enables the use of shorter probes that still have high melting temperatures

Probe is designed to have a higher melting temperature than the primers so it remains hybridized

Energy from the reporter is absorbed by the quencher but re-emitted as heat rather than light

Cleavage separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter

The increase in fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR

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66
Q

SYBR Green assay

A

Reaction chemistry: SYBR Green dye fluoresces when bound to dsDNA

Denaturation: When DNA is denatured, SYBR green dye is released, and fluorescence is reduced

Polymerization: PCR products are amplified

Polymerization complete: When polymerization is complete, dye binds to the dsDNA product resulting in a net increase in fluorescence

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67
Q

Compare 5’ nuclease assay and SYBR green assay

A

Nuclease:
Detects specific amplification products only

Specific hybridization between probe and target reduces false positives

A different probe has to be synthesized for each unique target sequence

SYBR Green:
Detects all amplified double stranded DNA including non-specific reaction products

Monitor the amplification of any double-stranded DNA sequences

No probes are required which reduces tour assay setup and running costs

Because SYBR green dye binds to any dsDNA including non-specific dsDNA sequences it may get false positive signals

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68
Q

Advantages of QPCR

A

The availability of commercial qPCR kits

Higher throughput and reduced user intervention

Automated set up, sample data analysis rapidly analyzed in software using the standard curve

Sensitive to same inhibitors faced in a traditional STR assay (both PCR based; amplifiable DNA?)

High sensitivity (down to a single copy number?)

Large dynamic range: ~30 pg to ~30 ng

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69
Q

QPCR disadvantages

A

qPCR is subject to inhibition, but IPC can help

<100 pg qPCR subject to variability and uncertainty

In highly degraded samples, assays that amplify short target sequences will detect and measure more DNA than assays that amplify long target sequences (relevant to STR typing)

Accurate quantitation assumes that each unknown sample is amplified at the same efficiency as the calibrant samples in the dilution series

70
Q

Quantifiler DUO DNA Quantification Kit

A

Designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample

71
Q

Target specific assays in Quantifiler kit

A

Two primers to amplify human DNA + One TaqMan probe labeled with VIC dye for detecting the amplified human target sequence

Two primers to amplify human male DNA + One TaqMan probe labeled with FAM dye for detecting the human male amplified target sequence

IPC:
Two primers to amplify a synthetic sequence not found in nature + One TaqMan probe labelled with NED dye for detecting the IPC DNA

72
Q

What are passive reference dyes

A

Passive reference dyes (ROX) used to normalize well-to-well fluorescence signal differences such as:

variation in the optical paths between wells

minor differences in volumes due to pipetting errors

73
Q

STR criteria for forensic applications

A

High discriminating power (i.e., high # of alleles)

High percentage of heterozygotes (e.g., HE > 70%) that amplify similarly well within/between individuals

Separate chromosomal locations to avoid linkage of loci (linkage would invalidate statistics of ‘product rule’ used)

Narrow allele range (100 - 400 bp) to amplify well in degraded DNA samples

Work in combination with other micros (multiplexing)
(Relatively) low mutation rate (i.e., frequencies do not change over time)

Low level of biological artifacts (i.e., stutter – more later!)

74
Q

Longest step in PCR process and how long does it take

A

Polymerase Chain reaction - 2.5-3 hours

75
Q

Advantages of PCR

A

Very small amounts of DNA template may be used from as little as a single cell

DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification

Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions

Contaminant DNA, such as fungal and bacterial sources will not amplify because human-specific primers are used

Commercial kits are now available for easy PCR reaction setup and amplification

76
Q

PCR disadvantages

A

The target DNA template may not amplify due to presence of PCR inhibitors in the extracted DNA

Amplification may fail due to sequential changes in the primer-binding region and the genomic DNA template

Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols

77
Q

How many cycles of PCCR are typically used

A

25-35

78
Q

What are stochastic effects of PCR

A

Stochastic sampling occurs with low template amounts, where unequal sampling of two alleles present in a heterozygous individual happens by chance

Results in allele imbalance, allele dropout, or complete locus dropout

Not an accurate reflection of the original DNA sample

79
Q

Oligonucleotide Primers

A

Primers must be specific to the target region, possess similar annealing temperatures, not interact significantly themselves (“hairpin structures”), or with each other (“primer dimers”)

80
Q

dNTPs

A

deoxyribonucleotide Triphosphates

Raw material for amplification (dATP, dCTP, dTTP, dGTP)

Used in excess to maximize efficiency of PCR

A high concentration of dNTPs can enhance reaction speed because they diffuse into the catalytic site of Taq DNA polymerase in a shorter amount of time

80
Q

Magnesium Ions

A

is a crucial factor affecting the performance of Taq and stringency of primer annealing

Cofactor in enzymatic reaction of DNA polymerization and without adequate free magnesium, Taq is inactive

Mg2+ neutralizes repulsion between negatively-charged DNA strands (template and primers), which has implications for primer binding stringency

81
Q

What is caused by low magnesium concentrations

A

repulsion of backbones of primer and template will be stronger (primer-template binding will be less stable); therefore, melting will occur at lower temperatures

82
Q

What is caused by high magnesium concentrations

A

neutralizes repulsion of primer and template backbones (primer-template binding more stable); therefore, melting will occur at higher temperatures

83
Q

What is optimal magensium concentration

A

but 1.5 mM concentration is standard, and it should never exceed 3.0 mM

84
Q

Explain banding patterns depending on magnesium concentrations

A

0.5: no banding

1.0: Minimal banding

1.5: Prime banding

> 2.0: Excess banding and drag

85
Q

Touchdown PCR

A

~50 cycles run at incrementally lower temperatures where the initial annealing temperature is higher than the primer melting temp, but gradually declines

First primer-template hybridization events are stringent, keeping undesired amplification low

86
Q

Hot start PCR

A

Taq polymerase has some activity below the optimum temp, so primers anneal non-specifically and create non-specific product at beginning of the run that will accumulate undesired, non-specific product.

In a hot start PCR, you add Taq at a higher temperature; therefore, it minimizes the effects of mis-priming!

87
Q

Hot start Taq

A

only start to work after an initial heat shock of 95C

pH decreases from 8.3 to 6.9 as the reaction buffer is affected by increase in heat (25oC to 95oC)

This pH change causes a modification to AmpliTaq Gold, and it is activated (called a Hot Start Taq)

Therefore, mis-priming occurs as reaction setup, but there is no accumulation of undesired, non-specific product

88
Q

Negative Control in PCR Components

A

Contents of the reaction mix and no DNA sample

You should never get PCR product (other than primer-dimers)

Product in your negative control indicates there must be contaminating DNA in one of your reagents

89
Q

Positive Control PCr components

A

Include one DNA sample that you know will work

If this sample fails to amplify, you know that there is something wrong with your reaction mix

The Identifiler STR genotyping kit we will use includes a positive control to evaluate the reaction efficiency

90
Q

Primer Control PCR components

A

Specific uses for human forensic biology labs

This control will tell you if it is due to your primers or because of one of your other reagents

91
Q

Electrophoresis

A

Goal: separate DNA by size due to negative charge

Electric field is applied such that the negatively charged DNA molecules migrate away from a negative towards a positive charge

smaller DNA molecules traveling faster than larger DNA molecules

92
Q

What does STR typing require (3)

A

Spatial resolution (separate STRs and loci)

Separate resolution (separate fluorescent dyes)

Sizing precision (run-run consistency; we use allelic ladders)

93
Q

2 types of gels used in electrophoresis

A

PAGE
Agarose

94
Q

PAGE gel

A

smaller pore size which resolves smaller DNA molecules (<1000 bp)

95
Q

Agarose Gel

A

larger size which resolves large DNA

96
Q

Use of formamide in PCR

A

Chemicals such as formamide or urea can be added to the DNA sample where they interact with DNA and interfere with the formation of hydrogen bonds between complementary ssDNA molecules

97
Q

Advantages of capillary electrophoresis

A

Fully automated, no need to individually load samples, don’t need to pour gels (electrokinetic injection of samples)

Only a subset of the amped sample used, can be re-tested.

Faster: higher voltages due to enhanced heat dissipation (300 V/cm vs 10 V/cm)

Electronic output: no need for gel pictures nor scanning gel.

No lane tracking, enclosed cap mea no lane bleed through, less cross-contamination

98
Q

Disadvantages to capillary electrophoresis

A

Each cap can only process one sample at a time, sequential injections (so 4, 16, or 96 caps).

Expensive to buy and maintain.

Salts, unwanted DNA can out-compete PCR products for sample injection

99
Q

STR genotypes vs STR profiles

A

An STR genotype is the allele (homozygote) or alleles (heterozygote) present for a particular locus.

An STR profile is preceded by combining all the STR genotypes (CODIS 13/20).

100
Q

Peak Detection threshold

A

Signal peak height is measured in relative fluorescence units (RFUs) that are related to the amount of DNA present in the sample loaded onto instrument

Detection thresholds typically vary from 50 RFU to 200 RFU

101
Q

Local Southern Method

A

DNA Fragment peaks are sized based on the curve produced from the points on the internal standard

102
Q

Off-ladder alleles

A

Defined as alleles that have a form of sequence variation compared to more commonly observed alleles

103
Q

Stutter Products

A

Peaks that show up one repeat unit less than the true allele due to strand slippage during DNA synthesis

Repeat unit bulges out when strand slippage occurs during replication

104
Q

Incomplete Adenylation

A

Taq DNA polymerase will often add an extra nucleotide to the end of a PCR product; most often an “A” (termed “adenylation”, or “A+”)

105
Q

Null Alleles

A

Allele is present in the DNA sample but fails to be amplified due to a nucleotide change in a primer binding site

Null alleles are a problem because a heterozygous sample appears falsely as a homozygote

106
Q

You perform a single-locus RFLP on locus D1S7 and after developing the Southern blot, you notice there is only one-band in the lane corresponding to suspect 1. The most likely explanation(s) for this result is:

A

suspect 1 is homozygous for locus D1S7

107
Q

The CODIS marker D7S820 is found within:

A

a noncoding region on chromosome 7

108
Q

The following treatments denature DNA from its double-stranded form:

A

high temperatures (>95°C)

109
Q

The term DNA fingerprinting arose after examining the results of which assay

A

single-locus RFLP

110
Q

Main advantages of multi-locus RFLPs are

A

the power to distinguish between individuals

the power to distinguish mixtures

111
Q

The Qiagen DNA extraction procedure we will use in the lab separates DNA from digested proteins using which of the following?

A

silica resin

112
Q

In DNA extraction protocols, what is the purpose of proteinase-K?

A

break peptide bonds in proteins to produce small peptides

113
Q

Where is the DNA after the centrifugation in step 6 after adding ethanol

A

Bound to the silica membrane

114
Q

Passive reference dye used in Quantifiler Duo DNA assay is

A

ROX

115
Q

SNP

A

Single nucleotide polymorphisms

a single-base sequence variation between individuals in an otherwise conserved region of DNA.

116
Q

What is the most common type of DNA sequence variation

A

SNPs are the most common type of DNA sequence variation. (90%)

117
Q

Advantages of SNPs in forensics

A

Short product - good marker when working with degraded dna

high multiplexing capabilities

easy to interpret results

118
Q

Disadvantages to SNPs in forensics

A

they are biallelic (C/T) only

Low variability means that 25-50 markers are required for the same power of discrimination

mixtures hard to interpret

119
Q

SNP Marker categories and applications

A

Identify SNPs, individual identification SNPs (IISNPs)

Lineage SNPs, lineage informative SNPs (LISNPs)

Ancestry SNPs, ancestry informative SNPs (AISNPs)

Phenotype SNPs, phenotype informative SNPs (PISNPs)

120
Q

IISNPs

A

Individual identification SNPs

SNPs that collectively give very low probabilities of two individuals having the same multi-locus genotype.

121
Q

LISNPs

A

Lineage information SNPs

sets of tightly linked SNPs that function as multi-allele markers that can serve to identify relatives with higher probabilities than simple bi-allelic SNPs.

122
Q

AISNPs

A

Ancestry information SNPS

SNPs that collectively give a high probability of an individual’s ancestry being from one part of the world or being derived from two or more areas of the world.

123
Q

PISNPS

A

Phenotype information SNPS

SNPs that provide a high probability that the individual has particular phenotypes, such as a particular skin colour, hair colour, eye colour etc.

124
Q

SNP analysis techniques

A

Reverse dot blot or linear arrays

Direct sequencing

Taqman 5/ nuclease assay

Pyrosequencing

125
Q

Reverse dot blot or linear arrays of SNPs

A

a series of allele-specific probes are attached to a nylon test strip at separate sites;

biotinylated PCR products hybridize to their complementary probes & are detected w a colorimetric reaction & evaluated visually.

126
Q

Direct sequencing of SNPs

A

products are sequenced and compared to reveal SNP sites.

127
Q

Taqman 5’ nuclease assay of SNPs

A

A fluorescent probe consisting of reporter and quencher dyes is added to a PCR reaction; amplification of a probe-specific product causes cleavage of the probe and generates an increase in fluorescence.

128
Q

Pyrosequencing of SNPs

A

Sequencing by synthesis of 20-30 nucleotides beyond primer sitel dNTPs are added in a specific order and those incorporated results in release of pyrophosphate and light through an enzyme cascade.

129
Q

Applications of SNPs to forensics

A

FDP - forensic DNA phenotyping - prediction of physical features

HIrisPlex System for prediction of hair and eye colour from DNA - hypothesised scenario for determination of genes

130
Q

Why will SNPs not replace sTRs for individual identification

A

Large databases with sTR profiles exist (need to replace data on existing samples with DNA markers)!

Mixture detection and interpretation benefits from marker systems with many alleles (most SNPs only have two alleles and three genotype possibilities).

Degraded DNA can be successfully analyzed by miniSTRs (thus removing one motivation for using SNPs, while maintaining compatibility with existing databases).

130
Q

Rules of inheritance for paternity testing

A

Child has two alleles for each autosomal marker (one from mom and one from biological father).

Child will have the mother’s mitochondrial DNA haplotype (barring mutation).

Child, if son, will have father’s Y-chromosome haplotype (barring mutation).

131
Q

What does kinship analysis require in paternity testing

A

The genotype at multiple loci for each of the individuals tested (eg; P,Q).

Relevant allele frequencies for the genetic loci examined (eg; from allele frequency tables).

A method to assess the relationship of interest based on genetic inheritance models (eg; paternity index or paternity exclusion).

132
Q

PI

A

Paternity index - likelihood ratio where:

Numerator assumes the individual is the father

Denominator assumes a random individual or person with similar ethnicity is the father.

133
Q

Types of lineage marker sand how they are passed down

A

Autosomal: genotype, passed on in part from all ancestors.

Y-Chromosome: haplotype,passed on complete but only by sons.

Mitochondrial: passed on complete but only by daughters.

Haplotype: the genetic type from a set of linked markers in an individual, such as that found with Y-STRs & mtDNA

134
Q

Benefits to Y-Chromosome testing in sexual assault evidence

A

male specific amplification, no need for differential extraction, determines # males in mixture

135
Q

Benefits to Y-Chromosome testing in paternity testing

A

tie to father without maternal profile, can use paternal relatives if father is unavailable.

136
Q

Advantages of Y chromosome DNA testing

A

Male specific amplification can enable examination of a male perpetrator’s profile even in mixtures with high levels of female DNA sexual assault cases.

Additional mixtures may possibly be analyzed (eg; fingernail scrapings, saliva on skin, etc).

Paternal transmissions from a father to all of his sons extends possible reference sample providers and enables tracing family lineages.

137
Q

Limitations of Y chromosome DNA testing

A

Since paternal relatives are identical, Y-STR typing cannot be used to distinguish among brother or even distant relatives.

the discrimination power of Y-STRs is limited by the size of the population database used.

Duplication and deletions can complicate the analysis.

138
Q

Characteristics of mtDNA

A

The “control region” (origin of replication) does not encode for gene products, therefore, fewer constraints on the control regions variability.

HV1 and HV2 are the focus of most forensic applications of mtDNA.

1-2% sequence variation bw unrelated individuals in these regions (~7-14 bp).

139
Q

Interpretation issues in mtDNA

A

Heteroplasmy: the presence of more than one mtDNA type in an individual

Mixtures

Nuclear Pseudogenes: some segments of the mtDNA genome have been inserted into the nuclear genome

140
Q

How can mRNA be used in presumptive testing to identify body fluid type?

A

Each cell in the human body has a unique pattern of gene expression

Some cells that will have genes that are on or off

Based on this we can identify body fluid type

141
Q

What is one advantage to using mRNA based presumptive testing in a forensic context

A

Advantages:
Highly specific
Highly sensitive
Simultaneous DNA isolation without loss of material

142
Q

What is the purpoose of the IPC in quantifiler duo results

A

Confirms negative results
Internal PCR Control

143
Q

What would your PIC be if you had inhibitors present?

A

High CT = PCR inhibitors or no amplification

144
Q

How doe presence of salt in your amplified DNA affect sample injection during a genotyping run? where does it come from and whats a solution

A

Competition a problem

Salt comes from extraction buffer and pcr

Solution is dilution in formamide

145
Q

Describe the two innovations that were used in the ABI identifiler kit to enable 16 loci to be simultaneously amplified and detected in a single reaction

A

Use more dyes when possible

Add non-nucleotide linkers to change mobility of PCR product which allows continued use of validated primers

Redesign primer and amplify varying amounts of flanking sequence

146
Q

Describe what stutter looks like on an electropherogram as it relates to RFUS and amplicon size

A

Repeat unit bulges out when strand breathing occurs during replication

Repeat unit depletion caused by slippage on the copied strand

Longer bp repeat motifs

147
Q

Describe why a matrix is required for genotyping, what the matrix is and why a matrix is essential when identifying peaks on a genotyping run

A

To reduce spectral overlap

Filters are set to detect emission spectra of each dye

Spectral overlap is removed by applying a matrix where samples labelled with a single dye are used to create a calibration file that shows spectral overlap between different dyes

148
Q

What does too little DNA in DNA quantification cause

A

Peak imbalance
ALlele dropout
Locus to locus imbalanc

149
Q

What does too much DNA cause in DNA quantification

A

Off scale peaks
Split peaks
Locus to locus imbalance

150
Q

What does each reaction contain in automated DNA sequencing

A

DNA template, Taq polymerase, primer, all dNTPS, and a small amount of the four dye-labeled dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP)

151
Q

Chelex extractions provide:

A

ssDNA

152
Q

In presumptive testing a what gene encodes a transcript that is ubiquitously expressed across all tissue types.

A

Housekeeping gene

153
Q

Features of FTA paper

A

lyses cells,
binds white blood cells to paper’s matrix,
protects DNA from nuclease activity,
and deters bacterial growth

154
Q

In DNA extraction protocols, what is the purpose of proteinase-K

A

break peptide bonds in proteins to produce small peptides

155
Q

The process of diluting or concentrating your DNA sample to an optimal range for the multiplex PCR is called

A

normalization

156
Q

At what stage of the qPCR do probes fluoresce

A

extension

157
Q

A mixture of primers that have sequence substitution of different bases but amplify the same locus are called

A

degenerate primers

158
Q

The goal of multicomponent analysis is to

A

Correct for spectral overlap

159
Q

What is the purpose of 9947A in the Identifiler assay

A

Used in the PCR for a positive control

160
Q

How are PCR products fluorescently labelled using the Identifiler Kit

A

using fluorescently labelled primers

161
Q

The sex-determining marker amplified in the Identifiler kit is the ______ locus

A

amelogenin

162
Q

The acronym “CODIS” stands for the ______

A

Combined DNA index system

163
Q

Which of the following agencies/labs cannot add DNA profiles generated from crime scene samples to the National DNA Data Bank

A

Federal bureau of investigation lab

164
Q

Which of the following types of information is not stored in CODIS for each sample

A

Brief criminal history

165
Q

If you were to discover that your sample was contaminated with extraneous human DNA, list two potential sources of this contamination.

A

sample-sample contamination
Contamination during amplification

166
Q

Describe two problems that occur when too much DNA is added to the PCR

A

causes fluorescence intensity to exceeds the linear dynamic range for detection by the instrument which means the quantitation for off-scale peaks is not accurate.
This can cause issues such as stutter and out of ladder results, causing less reliable and accurate results.
This can also lead to pull-up. This is when too much DNA is added to the intensity of the fluorescence of one dye, creating a peak in corresponding dye sequences as well, that do not represent alleles but are showing peaks because of the intensity.

167
Q

Describe two reasons why forensic labs are hesitant to perform PCR with >28 cycles

A

PCR cycle number is generally around 28-30 because PCR becomes “maxed out” due to exponential increase of products where reagents become limiting factors.
This means that the curve begins to plateau around 30 cycles. This can also lead to amplification of undesired regions.

168
Q

briefly describe the banding patterns you might expect to observe when you run your PCR products on an ethidium bromide stained agarose gel? Describe why you are observing this result on the gel by providing a description of the interaction of Mg2+ with primers and template DNA in the PCR.

A

The magnesium ion concentration is very low. This will cause the repulsion of the primer and template to be stronger, which causes instability. This causes melting to occur at lower temperatures. the banding patters will be almost non-existant since Taq is activated by magnesium ions, it will also not be activated.

169
Q

Define RMP and how can it be used in a forensic context

A

Random match probability can be used to express a DNA profile rarity estimate.
Therefore, random match probability can be defined as the frequency that the DNA profile would be observed in another unrelated individual from the same population.
It can be calculated by multiplying the frequencies of genotypes across all loci using the product rule. This can be used in a forensic contect in order to exclude or include suspects in a DNA comparison.
It does not prove guilt, it only provides information on the probability that the specific DNA profile would appear in a specified population

170
Q
A

Study Notes 3rd Year (10 decks)

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