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
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
3
Q
AFLP
A
Amplified fragment length polymorphism
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.
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
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.
7
Q
STR
A
Short tandem repeat
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
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
10
Q
What is CCT complex
A
CSF1PO
TPOX
TH01
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
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.
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
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
15
Q
Non-DNA based technology (2)
A
Blood group testing
Forensic protein profiling
16
Q
DNA based technology (4)
A
RFLP
PCR based (sequence)
PCR based (length)
Mitochondrial DNA sequencing
17
Q
DNA technology with low power of discrimination and slow speed of analysis
A
mtDNA
18
Q
DNA technology with low power of discrimination and fast speed of analysis
A
Polymarker D1S280 single STR
ABO blood groups
19
Q
DNA technology with high power discrimination and fast speed of analysis
A
Multiplex STRs
20
Q
DNA technology with high power discrimination and slow speed of analysis
A
RFLP multi-locus probes
RFLP single-locus probes
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
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
23
Q
Locus
A
Specific region of DNA
24
Q
Alleles
A
Alternative forms of a locus (A or a)
25
Homozygous
If two alleles are identical by descent at a locus
26
heterozygous
If two alleles are different by descent at a locus
27
Genotype
the characterization of alleles at a locus (AA or Bb)
28
DNA profile
the genotypes obtained at multiple loci (AABb)
29
How many chromosomes does a human have
22 pairs of chromosomes plus the XX or XY
30
DNA structure
includes sugar backbone, phosphate groups and four nucleotide bases
Phosphodiester bonds between adjacent nucleotides
31
What causes DNA to denature
Elevated temperatures or chemical treatments by disrupting hydrogen bonds i.e. formamide/salts
32
2 types of DNA polymorphisms
Sequence
Length
33
To be useful DNA markers must:
Variable (polymorphic)
Reproducible detection
34
Who developed DNA profiling
Alec Jefferys 1985
35
VNTR
Variable number of tandem repeats
Analyzed using RFLPs
36
3 possible outcomes of evidence examination
Exclusion - No match
Non-exclusion - Match or inclusion
Inconclusive - no result
37
Defense case in the OJ Simpson trial
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.
38
The basic rules of evidence collection and preservation
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
39
What are presumptive tests used for and what are some requirements
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
40
3 things presumptive tests, test for
Blood - serological methdods to detect hemoglobin or luminol
Semen - Serological methods to detect acid phosphatase or direct observation
Saliva - Serological methods to detect amylase
41
Advantages of body fluid identification using mRNA testing
high sensitivity
High specificity
simultaneous DNA isolation without loss of material
42
What DNA transcript can be used as a positive RNA control
"housekeeping genes"
43
Steps to DNA extraction
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!!
44
Goal of DNA extraction
maximize yield while minimizing contaminants/inhibitors from other cellular materials
45
Solution of Iron from RBC PCR inhibiton
Centrifugation
46
Solution of Ethanol PCR inhibitor
Heat elution with lid off
47
Solution to Minerals from bone PCR inhibitor
Decalcify prior to extraction
48
Solution to indigo dye, melanin, and protein PCR inhibitors
Dilution
49
What does increased hydrolytic cleavage of DNA due to high temperatures cause
nicks in the DNA tempate which can interfere with primer annealing or Taq polymerase
50
5 Extraction methods
Organic, Solid-phase (qiagen), Chelex, FTA paper, Differential extraction
51
Organic Extractions
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
52
Solid-phase DNA extraction
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
53
Qiagen Extraction
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
54
Promega Extraction
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
55
Differential extraction
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)
56
What does too much DNA in quantitation cause
Off-scale peaks
Split peaks (+/-A)
Locus-to-locus imbalance
Therefore you Must dilute sample
57
What does too little DNA in quantitation cause
Heterozygote peak imbalance
Allele drop-out
Locus-to-locus imbalance
Therefore you, Must concentrate sample
58
Slot Blot method
most used method during late 90s
Primate specific prove binds to satellite sequence
59
What wavelength do DNA and RNA absorb UV light
maximum of 260nm
60
What is considered a poor limit of detection
~3.5ng/ul
61
Pico Green Assay
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
62
What is the difference between PCr and Quantitative PCR
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
63
what is Ct
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
64
Two main types of Quantitative PCR assays
fluorogenic 5' nuclease
Inter-chelating dye SYBR green
65
5' Nuclease Assay
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
66
SYBR Green assay
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
67
Compare 5' nuclease assay and SYBR green assay
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
68
Advantages of QPCR
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
69
QPCR disadvantages
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
Quantifiler DUO DNA Quantification Kit
Designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample
71
Target specific assays in Quantifiler kit
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
What are passive reference dyes
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
STR criteria for forensic applications
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
Longest step in PCR process and how long does it take
Polymerase Chain reaction - 2.5-3 hours
75
Advantages of PCR
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
PCR disadvantages
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
How many cycles of PCCR are typically used
25-35
78
What are stochastic effects of PCR
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
Oligonucleotide Primers
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
dNTPs
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
Magnesium Ions
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
What is caused by low magnesium concentrations
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
What is caused by high magnesium concentrations
neutralizes repulsion of primer and template backbones (primer-template binding more stable); therefore, melting will occur at higher temperatures
83
What is optimal magensium concentration
but 1.5 mM concentration is standard, and it should never exceed 3.0 mM
84
Explain banding patterns depending on magnesium concentrations
0.5: no banding
1.0: Minimal banding
1.5: Prime banding
>2.0: Excess banding and drag
85
Touchdown PCR
~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
Hot start PCR
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
Hot start Taq
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
Negative Control in PCR Components
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
Positive Control PCr components
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
Primer Control PCR components
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
Electrophoresis
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
What does STR typing require (3)
Spatial resolution (separate STRs and loci)
Separate resolution (separate fluorescent dyes)
Sizing precision (run-run consistency; we use allelic ladders)
93
2 types of gels used in electrophoresis
PAGE
Agarose
94
PAGE gel
smaller pore size which resolves smaller DNA molecules (<1000 bp)
95
Agarose Gel
larger size which resolves large DNA
96
Use of formamide in PCR
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
Advantages of capillary electrophoresis
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
Disadvantages to capillary electrophoresis
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
STR genotypes vs STR profiles
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
Peak Detection threshold
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
Local Southern Method
DNA Fragment peaks are sized based on the curve produced from the points on the internal standard
102
Off-ladder alleles
Defined as alleles that have a form of sequence variation compared to more commonly observed alleles
103
Stutter Products
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
Incomplete Adenylation
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
Null Alleles
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
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:
suspect 1 is homozygous for locus D1S7
107
The CODIS marker D7S820 is found within:
a noncoding region on chromosome 7
108
The following treatments denature DNA from its double-stranded form:
high temperatures (>95°C)
109
The term DNA fingerprinting arose after examining the results of which assay
single-locus RFLP
110
Main advantages of multi-locus RFLPs are
the power to distinguish between individuals
the power to distinguish mixtures
111
The Qiagen DNA extraction procedure we will use in the lab separates DNA from digested proteins using which of the following?
silica resin
112
In DNA extraction protocols, what is the purpose of proteinase-K?
break peptide bonds in proteins to produce small peptides
113
Where is the DNA after the centrifugation in step 6 after adding ethanol
Bound to the silica membrane
114
Passive reference dye used in Quantifiler Duo DNA assay is
ROX
115
SNP
Single nucleotide polymorphisms
a single-base sequence variation between individuals in an otherwise conserved region of DNA.
116
What is the most common type of DNA sequence variation
SNPs are the most common type of DNA sequence variation. (90%)
117
Advantages of SNPs in forensics
Short product - good marker when working with degraded dna
high multiplexing capabilities
easy to interpret results
118
Disadvantages to SNPs in forensics
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
SNP Marker categories and applications
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
IISNPs
Individual identification SNPs
SNPs that collectively give very low probabilities of two individuals having the same multi-locus genotype.
121
LISNPs
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
AISNPs
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
PISNPS
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
SNP analysis techniques
Reverse dot blot or linear arrays
Direct sequencing
Taqman 5/ nuclease assay
Pyrosequencing
125
Reverse dot blot or linear arrays of SNPs
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
Direct sequencing of SNPs
products are sequenced and compared to reveal SNP sites.
127
Taqman 5' nuclease assay of SNPs
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
Pyrosequencing of SNPs
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
Applications of SNPs to forensics
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
Why will SNPs not replace sTRs for individual identification
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
Rules of inheritance for paternity testing
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
What does kinship analysis require in paternity testing
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
PI
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
Types of lineage marker sand how they are passed down
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
Benefits to Y-Chromosome testing in sexual assault evidence
male specific amplification, no need for differential extraction, determines # males in mixture
135
Benefits to Y-Chromosome testing in paternity testing
tie to father without maternal profile, can use paternal relatives if father is unavailable.
136
Advantages of Y chromosome DNA testing
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
Limitations of Y chromosome DNA testing
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
Characteristics of mtDNA
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
Interpretation issues in mtDNA
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
How can mRNA be used in presumptive testing to identify body fluid type?
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
What is one advantage to using mRNA based presumptive testing in a forensic context
Advantages:
Highly specific
Highly sensitive
Simultaneous DNA isolation without loss of material
142
What is the purpoose of the IPC in quantifiler duo results
Confirms negative results
Internal PCR Control
143
What would your PIC be if you had inhibitors present?
High CT = PCR inhibitors or no amplification
144
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
Competition a problem
Salt comes from extraction buffer and pcr
Solution is dilution in formamide
145
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
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
Describe what stutter looks like on an electropherogram as it relates to RFUS and amplicon size
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
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
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
What does too little DNA in DNA quantification cause
Peak imbalance
ALlele dropout
Locus to locus imbalanc
149
What does too much DNA cause in DNA quantification
Off scale peaks
Split peaks
Locus to locus imbalance
150
What does each reaction contain in automated DNA sequencing
DNA template, Taq polymerase, primer, all dNTPS, and a small amount of the four dye-labeled dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP)
151
Chelex extractions provide:
ssDNA
152
In presumptive testing a what gene encodes a transcript that is ubiquitously expressed across all tissue types.
Housekeeping gene
153
Features of FTA paper
lyses cells,
binds white blood cells to paper’s matrix,
protects DNA from nuclease activity,
and deters bacterial growth
154
In DNA extraction protocols, what is the purpose of proteinase-K
break peptide bonds in proteins to produce small peptides
155
The process of diluting or concentrating your DNA sample to an optimal range for the multiplex PCR is called
normalization
156
At what stage of the qPCR do probes fluoresce
extension
157
A mixture of primers that have sequence substitution of different bases but amplify the same locus are called
degenerate primers
158
The goal of multicomponent analysis is to
Correct for spectral overlap
159
What is the purpose of 9947A in the Identifiler assay
Used in the PCR for a positive control
160
How are PCR products fluorescently labelled using the Identifiler Kit
using fluorescently labelled primers
161
The sex-determining marker amplified in the Identifiler kit is the ______ locus
amelogenin
162
The acronym "CODIS" stands for the ______
Combined DNA index system
163
Which of the following agencies/labs cannot add DNA profiles generated from crime scene samples to the National DNA Data Bank
Federal bureau of investigation lab
164
Which of the following types of information is not stored in CODIS for each sample
Brief criminal history
165
If you were to discover that your sample was contaminated with extraneous human DNA, list two potential sources of this contamination.
sample-sample contamination
Contamination during amplification
166
Describe two problems that occur when too much DNA is added to the PCR
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
Describe two reasons why forensic labs are hesitant to perform PCR with >28 cycles
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
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.
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
Define RMP and how can it be used in a forensic context
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
