DNA Module 2

During developmental validations, what steps are assessed/performed to optimize the reaction?

"Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples." (SWGDAM, December 2012) Precision, accuracy, and reproducibility studies. Standard specimens (different tissues/fluids give same results), stutter percentages, characterization of loci, species specificity, sensitivity, stability, mixture studies, population studies (determine allele frequencies for population groups), environmental studies, matrix studies (different substrates).

SNP (single nucleotide polymorphism)

A single base sequence variation between individuals at a particular point in the genome is often referred to as a single nucleotide polymorphism or SNP

Advantages of STRs

*Easily amplified by the polymerase chain reaction without the problems of differential amplification (due to the fact that both alleles from a heterozygous individual are similar in size since the repeat size is small) *The number of repeats in STR markers can be highly variable among individuals, which make these STRs effective for human identification purposes. § PCR amplification of degraded DNA samples can be better accomplished with smaller product sizes. § Stutter products decrease with higher repeat number (like tetranucleotide) *Alleles not random but inherited from mom and dad so able to link to individual

Primer design parameters

*Primer length: 18-30 bases (too short then non-specific binding occurs, if too long higher probability for mutations and would require longer annealing time) *Similar annealing temps (multiplex so lots of locations) *Percentage GC content: 40-60% (if higher would need a higher temp to break bonds between primer and template) *No self-complementarity *No complementarity to other primer *Distance between two primers: less than 2000 bases apart *Sequence fairly conserved so limit primer binding mutations *No long runs with the same base (easier for mutations and errors to occur)

Mini STRs

-used specifically for LCN PCR and degraded samples -PCR products (=amplicons) designed to be as small as possible -smaller targets are more likely to remain intact in damaged/ degraded samples -Globalfiler has 10 mini STRs -Might have more alleles

Tetranucleotide

A sequence of four bases in a nucleic acid The four base spread in alleles with tetranucleotides makes closely spaced heterozygotes easier to resolve with size-based electrophoretic separations compared to alleles that could be two or three bases different in size with dinucleotides and trinucleotide markers, respectively.

What is a primer?

A short segment of DNA that acts as the starting point for a new strand

Other STR loci used in forensics?

Amelogenin, Y indel, DYS391, SE33, and Penta D

Thermal cyclers: how do they work?

An instrument used to carry out the PCR technique by subjecting specimens of DNA to programmed cycles of heating and cooling to specific temperatures for specific lengths of time. Heating and cooling done by element.

Advantages of multiplex PCR?

By adding more than one primer set to the reaction mixture, more than one region can be copied simultaneously. More information with less sample, cost effective, time saving, less input material required, etc.

13 core STR CODIS loci

CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11.

Cons of Y STR testing

Can't tell male profiles from same paternal lineage apart. Statistic for this testing is lower than STR testing since Y STRs are less polymorphic

When did the CODIS core loci increase to 20? How was this decision made and by whom? What are the additional loci?

Changed in 2017 to align with the FBI requirement made the same day. This date was based on a 2014 survey that indicated forensic labs would need about 18 months to make changes to their kit chemistry. Expanding core loci was talked about since 2011 to increase discrimination value and increase compatibility with international databases. Loci were chosen based on compatibility with existing loci (not on same chromosome or very far apart), vendor ability to incorporate, low mutations, highly discriminating, and no medical information at these loci.

Thermal cyclers: how are they maintained?

Checked quarterly according to manufacturer info (date of test, person doing test, if fails will be taken out of service): -Cycle performance test (sample block heating and cooling properly and PCR cycling function operates properly). -Heated cover verification (verify heated cover within + or - 3 Celsius of the 105C set point). -Temperature verification (verify average temp for sample block within + or - 0.25 Celsius of set point temp). -Temperature nonconformity test (Use this test to verify that the sample well temperatures are within the threshold temperature range) Thermal cycler temperature verification unit used to make sure those temperatures are accurate: temperature verification thermometer and probe

D1S1656

Chromosomal location: 1 CODIS 20

D10S1248

Chromosomal location: 10 CODIS 20

TH01

Chromosomal location: 11 CODIS 13

VWA

Chromosomal location: 12 CODIS 13

D12S391

Chromosomal location: 12 CODIS 20

D13S317

Chromosomal location: 13 CODIS 13

D16S539

Chromosomal location: 16 CODIS 13

D18S51

Chromosomal location: 18 CODIS 13

D19S433

Chromosomal location: 19 CODIS 20

D2S1338

Chromosomal location: 2 CODIS 20

D2S441

Chromosomal location: 2 CODIS 20

TPOX

Chromosomal location: 2 CODIS 13

D21S11

Chromosomal location: 21 CODIS 13

D22S1045

Chromosomal location: 22 CODIS 20

D3S1358

Chromosomal location: 3 CODIS 13

FGA

Chromosomal location: 4 CODIS 13

CSF1PO

Chromosomal location: 5 CODIS 13

D5S818

Chromosomal location: 5 CODIS 13

D7S820

Chromosomal location: 7 CODIS 13

D8S1179

Chromosomal location: 8 CODIS 13

Carryover contamination

Contamination of unamplified product by amplified product

cross-contamination

Contamination where DNA from one sample is introduced to another

Additional CODIS loci (to make 20)

D1S1656, D12S391, D10S1248, D2S1338, D22S1045, D19S433, D2S441

Internal vs. developmental validations vs. performance check

Developmental validation: the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic samples done by company Internal validation: an accumulation of test data created within the laboratory to demonstrate that established methods and procedures perform as expected in the laboratory Performance check: a quality assurance measure to assess the functionality of a laboratory critical equipment; may be used to describe a quality check (QAS) or a region-specific internal validation after an FDLE validation is performed (LAPM)

Replication in amp vs. in vivo

Different polymerases in vivo (only one type in PCR) Proofreading ability of polymerase in vivo (not in PCR) Replicate whole genome in vivo (specific target regions in PCR) Polymerase hot start, in vivo polymerase starts at origin of replication

Primers in vivo vs. in PCR reaction?

Fluorescent probe on primer used in PCR reaction, not on primers in vivo Primers in vivo are made from RNA but in PCR reaction are made from DNA Primers in PCR reaction lab created while primers in vivo are made by DNA polymerase alpha primase

Why use platinum Taq polymerase?

It is more advantageous compared to regular Taq because its hot start PCR enables us to produce results that are higher in specificity, sensitivity and yield. Non-specific products do not form during reaction set up at room temperature and a shorter amplification time. Don't have to add polymerase after incubation time.

Platinum Taq vs. Gold Taq

Gold: chemical hot start modification, 10-minute enzyme activation time, 60 sec/kb DNA synthesis speed, no inhibitor tolerance Platinum: antibody hot start modification, 2 minute enzyme activation time, 15 sec/kb DNA synthesis speed, inhibitor tolerance

Other applications of Y STR testing

Historical human migration patterns, genealogy, historical questions

Who discovered PCR?

Kary Mullis in 1985

What is meant by the term multiplexing?

Multiplex means multiple STRs can be examined in the same DNA test which produces highly discriminating results and allows for samples with degraded DNA to be analyzed.

What is a primer binding site mutation? What is its affect on amplification?

Mutation or sequence variation in primer binding site: Sequence variation does occur in the flanking regions surrounding STR loci, base change close to 5' end can destabilize primer annealing so amplification does not occur or reduces in efficiency to the point where alleles are not detectable (null alleles). If loci have primer binding site mutation can include multiple primers for that location to account for it.

Microvariant Alleles

Off-ladder occurs when allele does not line up with fragment sizes in the allelic ladder. Determine if truly off-ladder by comparing base pair sizes (determined by reference to the internal sizing standard) of the questioned sample and an allelic ladder marker under the same electrophoretic conditions.

SNP advantages

PCR products from SNPs can be less then 100bp in size, meaning these markers are able to withstand degraded DNA samples better than STRs (have amplicons 200-400 bps long) Can be multiplexed to higher level than STRs. Sample processing and data analysis more automated since size-based separation not needed. No stutter associated with each allele, helps simplify interpretation of the allele call May be able to predict ethnic origin and certain physical traits. Lower mutation rate than STR, more likely to be fixed in a population/ population specific = ethnic origin

Platinum Taq vs DNA polymerase in vivo

Platinum Taq can last 28 cycles in high heat due to its original source. It does not have proofreading ability but has a very low error rate. This should not affect analysis since at the end of PCR, there are millions of copies that have been made. The error that occurred would potentially be so small and enough of correct product would exist. As far as affecting the STRs, we look at length polymorphisms, not sequence. An incorrect base pairing in an STR would still have the correct length and would not expect to type differently.

Platinum Taq

Platinum taq is a component in the GF amplification kit that utilizes hot start PCR. Although made in the laboratory by the use of recombinant technology, it was derived from a thermophile called Thermus aquaticus, which is an aquatic bacterium found in hot springs at Yellowstone National Park. It is a chemically modified enzyme that contains a proprietary antibody, which blocks polymerase activity at room temperature. Temperature increase allows removal of the moiety. It also has the ability to add non-template adenosines (A's) to the 3' end product of each amplicon due to its terminal, transferase activity.

Lay term for PCR

Polymerase Chain Reaction Technique where make millions of copies of areas of DNA that testing for.

What is PCR?

Polymerase Chain Reaction; used to amplify DNA The polymerase chain reaction, which was first published by Kary Mullis in 1985, is an enzymatic process that is performed on a thermalcycler and utilizes synthetic amplification kits, such as Globalfiler. PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence. Involves heating and cooling samples in a precise thermal cycling pattern approximately 28-29 cycles.

Thermal cyclers: need for accuracy?

Precise and accurate sample heating and cooling is crucial to PCR in order to guarantee consistent results. Quick temperature changes make more accurate so no off-ladder amplification.

Heated lid of thermal cycler

Prevent condensation on samples. Make sure all liquid at bottom of reaction since dealing with very little volume so don't want to lose it.

SNP disadvantages

Three possible genotypes for SNPs, mixture interpretation can be difficult since can be hard to tell if true homozygote or mixture of two homozygotes or heterozygote and homozygote mixture Inability to simultaneously amplify enough SNPs in multiplexes from low amounts of DNA SNP by itself yields less info than STR marker, so must analyze a large number of SNPs in order to obtain power of discrimination to define a unique profile. (25-45 SNP needed for 13 core STRs and probs 50-100 needed for 10-16 STR loci)

What is amelogenin?

Sex typing gene that codes for a protein found in the developing tooth enamel Present in both the X and the Y chromosomes, but the X-linked version is shorter o Primers for this gene flank a 6bp deletion within intron 1 of the gene on the X homologue § 106bp from X chromosome and 112bp from Y chromosome

STRs

Short Tandem Repeats, regions of a DNA molecule that contain short segments consisting of three to seven repeating base pairs. Vary in length among individuals. o DNA markers used in forensic DNA typing o PCR-based and work with low-quantity DNA templates or degraded samples (easily amplified because heterozygous alleles similar in size since repeat size small) o STRs are highly discriminating between unrelated and even closely related individuals o Uses allele information instead of DNA sizes to compare results (easier to interpret) *Alleles not random but inherited Need more STRs than RFLPs to get same discrimination power (RFLPS more polymorphic)

Y STRs

Short tandem repeats located on the human Y chromosome. Their utility in forensic science is that they originate only from a male donor of DNA.

Thermal cycling parameters for currently used kits

Step 1: 95C for 1 min (initial incubation step) Step 2: 94C for 10 sec (denature) Step 3: 59C for 90 sec (anneal/extend), Repeat steps 2-3 for 29 cycles Step 4: 60C for 10 min (final extension), Step 5: 4C hold until taken off (up to 24 hours)

Stutter DNA product

Stutter products: small peaks several bases shorter than each STR allele peak · One repeat unit less than the corresponding main allele peak. This one is caused by deletion caused by forward slippage (one repeat more than is rare, caused by insertion by backward slippage). · Caused by slipped-strand mispairing (primer-template complex becomes unpaired during primer extension, allowing slippage of either primer or template strand such that one repeat forms a non-base-paired loop. Results in shortened PCR product that is less than the primary amplicon (STR allele) by a single repeat unit. Reduce stutter? Why we use loci with tetranucleotides because less occurrence, use different polymerases with less stutter percentages. Cannot stop stutter from occurring but can optimize reaction. The reason why we have stutter filters. Biological artifact

PCR inhibitors

Substances that interfere with DNA amplification in PCR (inhibit polymerase activity and can produce partial profiles). Mechanisms of inhibition: Interfere with cell lysis necessary for DNA extraction, interfere by nucleic acid degradation or capture, and inhibit polymerase activity which prevents enzymatic amplification of the target DNA. o Examples: textile dyes (indigo blue), hematin (heme), melanin, polysaccharides and bile salts from feces, leather, soil (humic compounds), urea in urine, and wood. Some reagents in DNA process can also inhibit PCR.

PCR components

Template DNA, primers, nucleotides, Taq polymerase, PCR controls, and other components (Tris-HCL, magnesium chloride, potassium chloride, bovine serum albumin or BSA) Master mix: enzyme, salts, dNTPs, bovine serum albumin, enzyme, buffer, and salt Primer set: forward and reverse primers

Hold step in PCR

The final hold step, at 4 deg, occurs where the samples are kept in a state similar to a refrigerator. They can be kept at this temperature for about 24 hours until ready for CE set up.

What is contamination?

The unintentional introduction of exogenous DNA into a sample or reaction

What do PCR components do?

Two primers: flank STR region and provide polymerase to bind and copy the template strand Template DNA: what is used to make copy of DNA Free DNTPs: building blocks that DNA polymerase uses to copy template DNA Taq polymerase: copies template DNA strand by binding to primer and adding free DTNPS TrisHCl/TE buffer: maintain pH at temps and raise volume to where it needs to be Magnesium chloride: Acts as cofactor to enhance activity of Taq Potassium chloride: Stabilizes primer-template binding Bovine serum albumin (BSA): minimize inhibition by stabilizing enzyme Sodium azide: preservative

Stochastic effects

When amplifying very low levels of DNA template, a phenomenon known as stochastic fluctuation can occur. Stochastic effects, which are an unequal sampling of the two alleles present from a heterozygous individual, result when only a few DNA molecules are used to initiate PCR. False homozygosity results if one of the alleles fails to be detected.

split peak/ partial adenylation

caused by unequal amplification due to too much of DNA template +A/-A mixture Partial adenylation where some PCR products have the extra adenosine (-A peaks) and some do (+A peaks) can contribute to peak broadness if the separation system resolution is poor (due to allele in one cycle got A and one didn't) Split peak = -A +A = A added to amplicon so same size

Environmental contamination

contamination of a sample from DNA in the environment (lab or analyst)

Degraded DNA

environmental exposure degrades DNA molecules by randomly breaking them into smaller pieces. § Some things that degrade DNA include water, and enzymes called nucleases that chew up DNA. Without an intact DNA strand that surrounds the STR repeat region to serve as a template strand, PCR will be unsuccessful because primer extension will halt at the break in the template.

differential amplification

is the selection of one target region or locus over another during the polymerase chain reaction. Differential amplification can also arise between two alleles within a single locus if one of the alleles has a mutation within a PCR primer-binding site, causing this allele to be copied less efficiently because of the primer-template mismatch. One locus amps better than others, smaller loci amplify better than others Biological artifact

Preferential amplification

is the unequal sampling of the two alleles present in a heterozygous locus primarily due to stochastic (random) fluctuation arising when only a few DNA molecules are used to initiate the polymerase chain reaction. Amplify one allele more than another Biological artifact

Anomaly (PCR)

non-reproducible, not observed consistently

Historical and current forensic DNA testing methods

o "DNA Fingerprinting" (RFLP) first described in 1985 by Alec Jeffreys (1986-2000) § Found certain regions of DNA contained repeated sections and that they differed from individual to individual (aka variable number of tandem repeats or VNTRs) § Used restriction fragment length polymorphism (RFLP) to examine VNTRs -high power of discrimination, slow speed of analysis -Requires more labor, time, and expertise -Restriction enzyme cuts at sites same in individuals, because longer repeats can only have a couple on a gel so have to do many gels which takes months *PCR 1985 Kary Mullis -Originally used water baths or incubators at each of the temps for PCR -Had to add polymerase after incubation, which involving opening reaction and potential for contamination *PM and DQ alpha (1990s): -Polymarker and DQ alpha, sequence polymorphisms -DQ alpha one location, few alleles -PM more locations but few alleles -Not multiplexing and not very discriminating -Time consuming, require more DNA (less than RFLP) -First PCR method in forensics o STR 2000-present § Short tandem repeats = high power of discrimination and rapid analysis speed § Smaller versions of VNTR sequences o mtDNA 1996-present § Low discrimination power and long sample processing time § Can be helpful in cases involving severely degraded DNA samples or when associating maternally related individuals *SNPs: Single nucleotide polymorphisms -Validation at FDLE in progress -Becoming more popular due to help in genetic genealogy (can tell how closely related individuals are to each other) -Need many more than STRs to be discriminating -Could potentially distinguish between identical twins (if look at enough locations) -Can get information for phenotypic traits *Mini STRs -FDLE used to offer this separately like Y STR but now global filer kit has them so don't do this anymore -Offered in Tallahassee -Primers closer to target region so smaller amplicon

Why were the original 13 core CODIS loci selected? How and by whom was this determined?

o Beginning in 1996, the FBI Laboratory sponsored a community-wide forensic science effort to establish core STR loci for inclusion within the national DNA database known as CODIS (Combined DNA Index System). Wanted these locations in order to make comparisons between labs easier and more standardized. § This STR Project beginning in April 1996 and concluding in November 1997 involved 22 DNA typing laboratories and the evaluation of 17 candidate STR loci. o At the STR Project meeting on 13-14 November 1997, 13 core STR loci were chosen to be the basis of the future CODIS national DNA database. The 13 CODIS core loci are CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. *Chosen because low mutations, highly discriminating, not on same chromosome (make sure inherited independently)

Non-forensic applications of PCR and STR technologies?

o Characterizing cell lines o Monitoring transplants (engraftment of donor cells after bone marrow transplants or stem cell transplants, help diagnose graft failure or relapse) o Detecting genetic chimeras (presence of two genetically distinct cell lines in an organism, can be acquired through blood stem cell transplants or blood transfusions or inherited o Monitoring needle sharing (differentiate between single and multi-person use of syringes) o Detecting cancer tumors (severe allelic imbalance at a locus in a single-source DNA sample almost appears as homozygote, genetic deletions common in tumors for many types of cancer) o Mapping genetic diseases o Examining human population density (ancestral origins of populations)

Mitochondrial DNA

o Good for degraded (because mtDNA is present in cells at a much higher copy number than the nuclear DNA from which STRs are amplified) or small amounts of DNA such as in bone, teeth, and hair o Due to high copy number more mtDNA molecules are more likely to survive than nuclear DNA. § High copy number means more risk of contamination o Makes up less than one percent of the total DNA content of a cell o For forensic and human identification purposes, human mitochondrial DNA is considered to be inherited strictly from one's mother.

Benefits of Y STR testing

o The Y chromosome is found only in males, and therefore genetic markers along the Y chromosome can be specific to the male portion of a male-female DNA mixture such as is common in sexual assault cases. o Y STR testing for sexual assault kits, male to female ratio (1:50) so drop at quant, forward to Y STR testing and isolate male DNA in sample Using Y chromosome specific PCR primers can improve the chances of detecting low levels of the perpetrator's DNA in a high background of the female victim's DNA. -Able to get Y STR results from males who are azospermic or vasectomized when concentrations of DNA do not meet regular STR thresholds.

PCR steps

o Three different temperatures that are repeated over and over 28 times and takes about 3 hours (5 min per cycle, 1 min per temp and 2 min between temps) Incubation: 95C for 1 min, allow DNA polymerase to become activated. temperature increase results in antibody that was blocking polymerase activity to become disassociated. Denaturation: 94C DNA strands separate or denature (10 seconds) Anneal: 59-60C forward and reverse primers bind or anneal to the DNA template and target region to be amplified (90 seconds) · Temperature is cooled to an appropriate temperature to bind (anneal) the oligonucleotide primers) Extension: 72C DNA polymerase extends the primers by copying the target region using deoxynucleotide triphosphate building blocks. Temperature is raised to the optimal temperature for DNA polymerase and it extends the primers to produce a copy of each DNA template strand (each DNA molecule doubles for each cycle). Final extension: 60C for 10 minutes, aka adenylation step. Adenosines added to ends of amplicons to make sure sharp peaks and more accurate allele calls Final hold step: 4C until samples are ready for CE set up (can be kept for about 24 hours here)

Artifact (PCR)

reproducible

Anomalies and artifacts associated with PCR

stochastic effect, stutter, differential amplification, preferential amplification, microvariant alleles, allele dropout/null allele, non-template addition

Importance of PCR

§ DNA from crime scenes is often limited in both quantity and quality and obtaining a cleaner, more concentrated sample is normally out of the question (most perpetrators of crimes are not surprisingly unwilling to donate more evidence material). The PCR DNA amplification technology is well suited to analysis of forensic DNA samples because it is sensitive, rapid, and not limited by the quality of the DNA as the restriction fragment length polymorphism (RFLP) methods are.

Non-template addition in PCR

§ DNA polymerases, particularly the Taq polymerase used in PCR, often add an extra nucleotide to the 3′-end of a PCR product as they are copying the template strand § Most often adenosine and is sometimes referred to as adenylation or +A form of the amplicon § Non-template addition results in a PCR product that is one base pair longer than the actual target sequence.

How to overcome PCR inhibitors?

§ Diluting DNA template, also dilutes PCR inhibitor, so that re-amplified in the presence of less inhibitor. (results in polymerase binding to inhibitor and remove from reaction so rest of polymerase can amplify DNA). § BSA or betaine have been shown to prevent or minimize the inhibition of PCR. Sodium hydroxide treatment of DNA has been shown to neutralize inhibitors of polymerase. Aluminum ammonium sulfate helps prevent inhibitors in soil. § Microcon filters have been used to separate extracted DNA from inhibiting compounds

Contamination prevention

§ Pre- and post-PCR sample processing areas should be physically separated. Usually a separate room or a containment cabinet is used for setting up the PCR amplification reactions. § Equipment, such as pipettors, and reagents for setting up PCR should be kept separate from other laboratory supplies, especially those used for analysis of PCR products. § Disposable gloves should be worn and changed frequently § Reactions may be set up in laminar hood § Aerosol-resistant pipette tips should be used and changed on every new sample to prevent cross-contamination during liquid transfers § Reagents should be carefully prepared to avoid presence of contaminating DNA § Clean workspaces and instruments with bleach solutions and isopropanol to insure extraneous DNA destroyed prior to extraction or PCR

Purpose of non-template addition in PCR?

§ Sharper peaks improve the likelihood that a system's genotyping software can make accurate calls. In addition, variation in the adenylation status of an allele across multiple samples can have an impact on accurate sizing and genotyping potential microvariants. For example, a non-adenylated TH01 10 allele would be the same size as a fully adenylated TH01 9.3 allele because they contain an identical number of base pairs. Therefore, it is beneficial if all PCR products for a particular amplification are either +A or −A rather than a mixture of +/−A products.

tri-alleles

§ These extra peaks are not a result of a mixture but are reproducible artifacts of the sample. Extra chromosomal occurrences or primer point mutations have been known to happen and result in a three banded pattern. · Three peaks equal in intensity most likely means tri-allele and not a mixture, especially if other locations have two peaks with similar height ratios. (probs chromosome duplication) · Trisomy 21 down syndrome Biological artifact

Allele Dropout/Null allele

· Allele dropout may occur due to mutations (variants) at or near the 3′-end of a primer and thus produce little or no extension during PCR. · If a base pair change occurs in the DNA template at the PCR primer binding region, the hybridization of the primer can be disrupted resulting in a failure to amplify, and therefore failure to detect an allele that exists in the template DNA. Biological artifact

Disadvantages of PCR

· The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA · Amplification may fail due to sequence changes in the primer-binding region of 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

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.