Forensic Biology Chapter 7

thermal cycler

The instrument that heats and cools a DNA sample in order to perform the PCR reaction is known as a _____________ _________.

Kary Mullis

The inventor of PCR is _________ ____________.

amplification

The longest single step in this process is multiplex PCR ______________, which requires approximately 3 hours using manufacturer-supplied protocols for commercial STR typing kits.

0.2

The most common tube in use with 20- to 50- μ L PCR reactions is a thin-walled _______-mL tube.

Taq

The most commonly used thermal stable polymerase is ________, which comes from a bacterium named Thermus aquaticus that inhabits hot springs.

buffer

The negative control usually contains water or __________ of the same volume as the DNA template.

Tris

The pH of the __________ buffer in the PCR reaction varies with temperature; higher temperatures cause the solution pH to go down by approximately 0.02 pH units with every 1 ° C.

adding

The polymerase chain reaction permits more than one region to be copied simultaneously by simply __________ more than one primer set to the reaction mixture.

blank

An extraction ' _________ ' is also useful to verify that the reagents used for DNA extraction are clean of any extraneous DNA templates.

7.0

At a pH below ________ the chemical modification moieties fall off and the activity of the polymerase is restored.

accurate

At such low volumes, evaporation can be a problem and ___________ pipetting of the reaction components can become a challenge.

Primer dimers are a particular problem. Why?

Because their small size relative to the PCR products means that they will be preferentially amplified.

carryover

PCR product _____________ results from amplified DNA contaminating a sample that has not yet been amplified.

peak heights

PCR product yields in the form of STR ________ ____________ produced by a commercial kit might be evaluated at the optimal annealing temperature (e.g., 59 ° C) and 2 ° and 4 ° C higher and lower (e.g., 55 ° , 57 ° , 61 ° , and 63 ° C). Differences, if any, would then be noted relative to the optimal annealing temperature supplied in the kit protocol.

dropout

PCR reactions involving DNA template levels below approximately 100 pg of DNA, or about 15 diploid copies of genomic DNA, have been shown to exhibit allele ____________.

contamination

The sensitivity of PCR necessitates constant vigilance on the part of the laboratory staff to ensure that ______________ does not affect DNA typing results.

multiplex

The simultaneous amplification of two or more regions of DNA is commonly known as multiplexing or ____________ PCR.

position

The target region on the DNA template is defined by the _____________ of the primers.

28, 32

The time required for ____ to ____ cycles is typically 2.5 to 3 hours.

The most important components of a PCR reaction are:

The two primers, which are short DNA sequences that precede or 'flank ' the region to be copied.

cycler

There are a wide variety of thermal _________ options available from multiple manufacturers.

avoided

The primer annealing temperatures should be similar and excessive regions of complementarity should be ___________ to prevent the formation of primer dimers that will cause the primers to bind to one another instead of the template DNA.

Disadvantages of the Hot-Start approach:

1) It is cumbersome and time consuming when working with large numbers of samples 2) the sample tubes must be opened at the thermal cycler to introduce the essential component, which gives rise to a greater opportunity for cross-contamination between samples.

knowledge

Some ______________ of the DNA sequence to be copied is required in order to select appropriate primer sequences.

Inhibitors can:

1) (1) interfere with the cell lysis necessary for DNA extraction 2) interfere by nucleic acid degradation or capture 3) inhibit polymerase activity, thus preventing enzymatic amplification of the target DNA

sources of contamination:

1) A scientist setting up the PCR reaction can inadvertently add his or her own DNA to the reaction if he or she is not careful. 2) the police officer or crime scene technician collecting the evidence can contaminate the sample if proper care is not taken.

The phi29 enzyme has a high processivity and can amplify fragments of up to 100 kb. Why?

1) Because it displaces downstream product strands, enabling multiple concurrent and overlapping rounds of amplification. 2) It has a higher replication fidelity compared to Taq polymerase due to 3 ́ - 5 ́ proofreading activity.

With Primer 3, the user inputs:

1) DNA sequence 2) specifies the target region within that sequence to be amplified. 3) PCR product size 4) primer length 5) desired annealing temperature

To avoid contamination:

1) Each piece of evidence should be collected with clean tweezers or handled with disposable gloves that are changed frequently. 2) Laboratory personnel should be appropriately gowned during interactions with samples prior to PCR amplification. 3) The appropriate covering includes lab coats and gloves as well as facial masks and hairnets to prevent skin cells or hair from falling into the amplification tubes.

DNA amplification process with the polymerase chain reaction:

1) In each cycle, the two DNA template strands are first separated (denatured) by heat. 2) The sample is then cooled to an appropriate temperature to bind (anneal) the oligonucleotide primers. 3) Finally the temperature of the sample is raised to the optimal temperature for the DNA polymerase and it extends the primers to produce a copy of each DNA template strand. ---For each cycle, the number of DNA molecular fragments (with the sequence between the two PCR primers) double.

Tips for avoiding contamination with PCR reactions in a laboratory setting include:

1) 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. 2) 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. 3) Disposable gloves should be worn and changed frequently. 4) Reactions may also be set up in a laminar fl ow hood, if available. 5) Aerosol-resistant pipette tips should be used and changed on every new sample to prevent cross-contamination during liquid transfers. 6) Reagents should be carefully prepared to avoid the presence of any contaminating DNA or nucleases. 7) Ultraviolet irradiation of laboratory PCR setup space when the area is not in use and cleaning workspaces and instruments with isopropanol and/or 10% bleach solutions help to ensure that extraneous DNA molecules are destroyed prior to DNA extraction or PCR setup.

A number of primer design software packages are commercially available, including:

1) Primer Express (Applied Biosystems, Foster City, CA) 2) Oligo (Molecular Biology Insights, Cascade, CO)

Additional challenges of Primer design for the STR DNA markers include:

1) Primers need to be adjusted on the STR markers to achieve good size separation between loci labeled with the same fluorescent dye. 2) Primers must produce robust amplifications with good peak height balance between loci as well as specific amplification with no nonspecific products that might interfere with proper interpretation of a sample's DNA profile. 3) Primers should produce a maximal non- template-dependent ' ' +A ' addition to all PCR products to ease data interpretation.

Problems with WGA:

1) Stochastic effects at low levels of DNA template prevent WGA from working reliably. 2) Allele dropouts from STR loci were observed at 50- and 5-pg levels of starting material just as are seen with current low copy number DNA testing.

Reasons why the PCR DNA amplification technology is well suited to analysis of forensic DNA samples:

1) because it is sensitive 2) it is rapid 3) it is not limited by the quality of the DNA as are the restriction fragment length polymorphism (RFLP) methods.

When setting up a set of samples that contain the same primers and reaction components, it is common to prepare a ' master mix ' that can then be dispensed in equal quantities to each PCR tube. Why?

1) helps to ensure relative homogeneity among samples 2) by setting up a larger number of reactions at once, small pipetting volumes can be avoided, which improves the accuracy of adding each component (and thus the reproducibility of one's method)

PCR inhibitors and their sources:

1) inhibitor: Heme (hematin); source: Blood 2) inhibitor: Melanin; source: Tissue and hair 3) inhibitor: Polysaccharides; source: Feces 4) inhibitor: Bile salts; source: Feces 5) inhibitor: Humic compounds; source: Soil 6) inhibitor: Urea; source: Urine 7) inhibitor: Textile dyes (denim); source: Clothing (blue jeans)

Thermal cyclers vary in:

1) the number of samples that can be handled at a time 2) the size of the sample tube 3) the volume of reagents that can be handled 4) the speed at which the temperature can be changed

For the PCR to work efficiently:

1) the two primers must be specific to the target region 2) must possess similar annealing temperatures 3) must not interact significantly with each other or themselves to form ' primer dimers ' 4) must be structurally compatible 5) the sequence region to which the primers bind must be fairly well conserved because if the sequence changes from one DNA template to the next, then the primers will not bind appropriately

Components of a PCR reaction consist of:

1) two primers 2) template DNA that will be copied 3) dNTP building blocks that supply each of the four nucleotides, 4) a DNA polymerase that adds the building blocks in the proper order based on the template DNA sequence

undesirable

Once low-temperature nonspecific priming occurs, these ______________ products will be efficiently amplified throughout the remaining PCR cycles.

positive

A ' __________ control ' is a valuable indicator of whether or not any of the PCR components have failed or were not added during the reaction setup phase of experiments conducted.

adenylation

A 30- to 60-minute 60 ° C soak is used at the end of thermal cycling to enable full _____________ of the PCR products produced.

deionized

A PCR reaction is prepared by mixing several individual components and then adding _____________ water to achieve the desired volume and concentration of each of the components.

8.3

A Tris buffer with pH ______ at 25 ° C will go down to pH ~6.9 at 95 ° C.

primer

A __________ is a chemically synthesized oligonucleotide that is added in a high concentration relative to the DNA template to drive the PCR reaction.

recovery

A common challenge with forensic casework is the ___________ of limited quantities of DNA from evidentiary samples.

The result of amplifying a DNA sample containing an inhibitor such as hematin is:

A loss of the alleles from the larger sized STR loci or even complete failure of all loci.

closed

A modified form of Taq DNA polymerase has been developed that requires thermal activation and thus enables _________-tube hot-start PCR.

same

A multiplex PCR makes use of two or more primer sets within the __________ reaction mix.

300

A nanogram of human genomic DNA contains only about _______ copies of single-copy DNA markers.

whole genome

A new DNA enrichment technology has been developed known as _________ ____________ amplification (WGA).

target

A primer acts to identify or ' __________ ' the portion of the DNA template to be copied.

Primer 3

A primer design program called ___________ ____ is available on the World Wide Web through the Whitehead Institute (http://frodo.wi.mit.edu/).

template

A standard DNA ___________ of known sequence with good-quality DNA should be used for the positive control.

evaluation

A thorough _______________ of performance for a multiplex will also involve addition and removal of primer sets to see if overall balance in other amplification targets are affected.

molecular biology

A wide range of PCR cycling protocols have been used for various ___________ ___________ applications.

prevent

Additives to PCR reactions, such as bovine serum albumin (BSA), have been shown to ____________ or reduce PCR inhibition.

optimization

Each new PCR application is likely to require some degree of _________________ in either the reagent components or thermal cycling conditions. Multiplex PCR is no exception.

pipet

Although it is challenging to _________ the small volumes necessary to perform this work, this technique is cost effective due to reduced reagent consumption.

modified

AmpliTaq Gold ™ DNA polymerase is a chemically ___________ enzyme that is rendered inactive until heated.

preincubation

An extended _____________ of 95 ° C, usually for 10 or 11 minutes, is used to activate the AmpliTaq Gold.

Larger solution volumes lead to thermal equilibrium issues for the reaction mixture. Why?

Because it takes longer for an external temperature change to be transmitted to the center of a larger solution than a smaller one.

Liquid is attracted to the defined positions and forms a small bead, which can then be covered with 5 μ L of mineral oil before thermal cycling is performed in order to prevent evaporation. Why is the liquid attracted?

Because of the chemical nature of the hydrophilic spots.

Multiplex PCR optimization is more of a challenge than singleplex reactions. Why?

Because so many primer-annealing events must occur simultaneously without interfering with each other.

preferentially

Because the amplified DNA is many times more concentrated than the unamplified DNA template, it will be _______________ copied during PCR and the unamplified sample will be masked.

simplified

Commercial kits with premixed components have greatly _____________ the use of PCR in forensic DNA laboratories.

relative

Concentrations of magnesium chloride and deoxynucleotide triphosphates are typically increased slightly ______________ to singleplex reactions.

sensitive

Contamination of PCR reactions is always a concern because the technique is very _____________ to low amounts of DNA.

minuscule

Contamination prevention precautions are especially critical when working with ______________ amounts of sample or sample that has been degraded.

negative

Controls typically include a ' ____________ control, ' which is the entire PCR reaction mixture without any DNA template.

Number of target DNA molecules created by PCR amplification (if reaction is working at 100% efficiency):

Cycle Number Number of Double- Stranded Target Molecules (Specific PCR Product) 1 0 2 0 3 2 4 4 5 8 6 16 7 32 8 64 9 128 10 256 11 512 12 1024 13 2048 14 4096 15 8192 16 16,384 17 32,768 18 65,536 19 131,072 20 262,144 ----Number keeps doubling with each cycle. Usually goes to 34 cycles.

components

Cycling parameters differ because reaction _______________, in particular the primer concentrations and sequences, vary between the different manufacturers ' kits.

limited

DNA from crime scenes is often ___________ 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 to aid in their prosecution).

The primary reason that PCR protocols vary is:

Different primer sequences have different hybridization properties and thus anneal to the DNA template strands at different rates.

copy

During each PCR cycle, a _________ of the target DNA sequence is generated for every molecule containing the target sequence.

anneal

During thermal cycling, at 60 ° C, primers bind or ' ___________ ' to the DNA template and target the region to be amplified.

extends

During thermal cycling, at 72 ° C, the DNA polymerase ____________ the primers by copying the target region using the deoxynucleotide triphosphate building blocks.

denature

During thermal cycling, at 94 ° C, the DNA strands separate, or ' ______________. '

anchor

Each ' __________ ' spot on this AmpliGrid holds 1 μ L of solution containing the reaction components necessary for PCR.

low copy number

Efforts have been made to obtain results with ______ _________ ___________ (LCN) DNA testing.

millions

Even a tiny droplet of PCR product left in a pipette tip may contain __________ of copies of the amplifiable sequence.

balance

Extensive optimization is normally required to obtain a good ______________ between the amplicons of the various loci being amplified.

homozygosity

False _____________ results if one of the alleles fails to be detected.

1985

First described in _________ by Kary Mullis and members of the Human Genetics group at the Cetus Corporation (now Roche Molecular Systems), PCR has revolutionized molecular biology with the ability to make hundreds of millions of copies of a specific sequence of DNA in a matter of only a few hours.

compatible

For a multiplex reaction to work properly, the primer pairs need to be _________________.

480

For a number of years there were some forensic laboratories that used an older model thermal cycler called the DNA Thermal Cycler ________.

low

Forensic DNA specimens often possess _______ levels of DNA.

polymerase

Forensic science and DNA typing laboratories have greatly benefited from the discovery of a technique known as the ______________ chain reaction, or PCR.

profiles

Full DNA ___________ down to 32 pg of DNA template have been achieved with a commercial STR typing kit.

redesign

Obtaining successful coamplification with well-balanced PCR product yields sometimes requires _____________ of primers and tedious experiments with adjusting primer concentrations.

textile dyes, hemoglobin

Occasionally substances such as _____________ ________ from clothing or ________________ from red blood cells can remain with the DNA throughout the sample preparation process and interfere with the polymerase to prevent successful PCR amplification.

reaction component

Hot-start PCR may be performed by introducing a critical ___________ _____________, such as the polymerase, after the temperature of the sample has been raised above the desired annealing temperature (e.g., 60 ° C).

32

In an ideal situation, after _____ cycles approximately a billion copies of the target region on the DNA template have been generated.

96, 384

In higher throughput labs, _____-well or ______-well plates are routinely used for PCR amplification.

performance

Internal validation studies focus on _____________ of the multiplex with varying conditions around the optimal parameters supplied with the kit protocol.

dimers

It is also possible at a low temperature for the primers to bind to each other, creating products called ' primer __________.

hold times

Longer __________ ___________ are needed at each temperature, which leads to longer over-all thermal cycling times.

hot-start

Low-temperature mispriming can be avoided by initiating PCR at an elevated temperature, a process usually referred to as ' ______-_________' PCR.

isothermal

MDA is ____________ with an incubation temperature of 30 ° C and requires no heating and cooling like PCR.

A critical reaction component:

Minimizes the possibility of mispriming and misextension events by not having the polymerase present during reaction setup.

condensing

Modern thermal cyclers use a heated lid to keep the PCR reagents from ________________ at the top of the tube (or sealed plate sample well) during the temperature cycling.

protocols

Most molecular biology _____________ for PCR are thus in the 20- to 50- μL range.

mispriming

Not only is the template DNA well denatured but the polymerase is activated just when it is needed, and not in a situation where primer dimers and ____________ can occur as easily.

challenging

Obtaining a nicely balanced multiplex PCR reaction with each PCR product having a similar yield is a ______________ task.

shorter

Other DNA polymerases have been developed recently that are rendered active for hot-start PCR with __________ times (e.g., 1 - 2 minutes instead of 10 - 11 minutes). These enzymes can help reduce the overall time for PCR amplification and enable rapid PCR efforts.

yield

PCR __________ is directly affected by the annealing characteristics of the primers.

premade

PCR has been simplified in recent years by the availability of reagent kits that allow a forensic DNA laboratory to simply add a DNA template to a ____________ PCR mix containing all the necessary components for the amplification reaction.

purification

PCR inhibitors may be removed during DNA extraction or through additional DNA _______________ steps.

5, 100

PCR is commonly performed with a sample volume in the range of ___ to _____ μ L.

10

Part of the 2.5 - 3 hour cycle run time is a _____-minute front-end hot start to activate the AmpliTaq Gold DNA polymerase.

GeneAmp

Perhaps the most prevalent thermal cycler in forensic DNA laboratories is the _____________ 9700 from Applied Biosystems (Foster City, CA).

reused

Pipette tips should never be ____________.

wells

Plastic ' plates ' containing 96 _______ can be used that are covered or sealed once the PCR reaction mix has been added.

heating, cooling

Precise and accurate sample __________ and __________ are crucial to PCR in order to guarantee consistent results.

research

Premade PCR kits are optimized through extensive ______________ efforts on the part of commercial manufacturers.

aliquot

Premade PCR kits are typically prepared so that a user adds an __________ of the kit solution to a particular amount of genomic DNA.

10,000

Prices for thermal cycling devices range from a few thousand dollars to more than $___________.

singleplex

Primer 3 works well for quickly designing _____________ primer pairs that amplify just one region of DNA at a time.

concentrations

Primer _________________ are one of the largest factors in a multiplex PCR reaction determining the overall yield of each amplicon.

nearest neighbor

Primer design programs use thermodynamic ' ____________ ___________ ' calculations to predict annealing temperatures and primer interactions with themselves or other possible primers.

magnesium

Primer sequences and concentrations along with ______________ concentrations are usually the most crucial to multiplex PCR.

nonspecifically

Primers can anneal _______________ to the template DNA at room temperature when PCR reactions are being set up and nonspecific products may result.

cocktails

Qiagen (Valencia, CA) and Sigma-Aldrich (St. Louis, MO) both offer phi29 DNA polymerase _______________ for performing WGA. The kit sold by Qiagen is called REPLI-g while Sigma- Aldrich's kit is GenomePlex.

72

Regular DNA polymerases exhibit some activity below their optimal temperature, which for Taq polymerase is _____ ° C.

titrations

Repeated experiments and primer ______________ are usually performed to achieve an optimal balance.

ramp

STR kit manufacturers have validated their kits with 1 ° C/ second temperature _________ rates and dwell times of around 1 minute per temperature step.

mineral oil

Samples amplified in the 480 required an overlay of a drop of ___________ _____ on top of the PCR reaction mix in order to prevent evaporation since there is no heated lid.

20

Stochastic artifacts can be avoided by adjusting the cycle number of the PCR reaction such that approximately ______ or more copies of target DNA are required to yield a successful typing result.

interior

Textile dyes, leather, and wood surfaces from ____________ crime scenes may also contain DNA polymerase inhibitors.

amplified

The DNA template should be _____________ with the same PCR primers that are used on the rest of the samples in the batch that is being ______________.

768

The Dual 384-well GeneAmp ® PCR System 9700 can run ________ reactions simultaneously on two 384-well sample blocks.

96

The GeneAmp 9700 can heat and cool _____ samples in an 8 X 12-well microplate format at a rate of approximately 1 ° C per second.

0.2 mL

The GeneAmp 9700 uses ______ _______ tubes with tube caps. These tubes may be attached together in strips of 8 or 12, in which case they are referred to as ' strip-tubes '.

Primer-Blast

The National Center of Biotechnology Information recently released a combined __________-__________ tool that enables finding specific PCR primers through combining the features of Primer3 and BLAST (see http://www.ncbi .nlm.nih.gov ).

inhibitors

The PCR amplification process can be affected by substances known as ' _____________, ' which interfere or prevent the DNA amplification process from occurring properly.

multiple displacement

The WGA enzymes work by __________ ______________ amplification (MDA), which is some- times referred to as rolling circle amplification.

Internet

The ___________ has become a valuable resource for tools that aid primer selection.

addition

The ____________ of each new primer in a multiplex PCR reaction exponentially increases the complexity of possible primer interactions.

development

The _______________ of an efficient multiplex PCR reaction requires careful planning and numerous tests and efforts in the area of primer design and balancing reaction components.

inadvertent

The _______________ transfer of even a very small volume of a completed PCR amplification to an unamplified DNA sample can result in the amplification and detection of the ' contaminating ' sequence.

biometric

The ability to perform multiplex PCR amplification with commonly used STR markers in a few minutes rather than hours could open new potential _____________ applications for DNA testing including analysis of individuals while they wait at a country border or an airport.

5

The availability of ___-dye detection systems has enabled development of multiplexes capable of amplifying and analyzing in excess of 20 short tandem repeat loci.

corresponds

The best results with premade PCR commercial kits are obtained if the DNA template is added in an amount that _____________ to the concentration range designed for the kit.

primers

The boundaries of the amplified product are defined by oligonucleotide ____________ that are complementary to the 3 ́ ends of the sequence of interest.

lysine

The chemical modification involves a derivitization of the epsilon-amino groups of the __________ residues.

8, 10

The current forensic DNA typing process takes about ____ to ______ hours.

3 hours

The entire PCR process is about ___ _________ in duration with each cycle taking ~5 minutes on conventional thermal cyclers: 1 minute each at 94° , 60° , and 72° C, and about 2 minutes ramping between the three temperatures.

in situ

The entire chip is then placed in a thermal cycler with an _____ _______ adapter for the PCR heating and cooling step.

AmpliTaq Gold

The enzyme, ____________ ________, has greatly benefited the specificity of PCR amplifications.

laboratory

The evidence samples are typically processed through a forensic DNA ______________ prior to the suspect reference samples to avoid any possibility of contaminating the evidence with the suspect's amplified DNA.

Nobel Prize

The impact of PCR has been such that its inventor, Kary Mullis, received the _________ _________ in Chemistry in 1993 — less than 10 years after it was first described.

reaction

The sample is pipetted into a variety of ___________ tubes designed for use in PCR thermal cyclers.

portable

There is great interest in developing ____________, rapid DNA typing devices for a number of applications.

384

Thermal cyclers capable of amplifying ______ samples or more at one time are now available.

valuable

Thermal cyclers capable of high sample volume processing are ____________ in production settings but are not widely used in forensic DNA laboratories.

3

Thermal cycling typically involves ____ different temperatures that are repeated over and over again 28 to 32 times.

strip-tubes

These 0.2-mL tubes can be purchased as individual tubes with and without attached caps or as ' ________-_________ ' with 8 or 12 tubes connected together in a row.

crime scenes

These inhibitors can be present in DNA samples collected from __________ ____________.

Why are controls useful?

They are useful for assessing whether or not any of the PCR components have been contaminated by DNA (e.g., by you or someone else in your lab).

amplicon

This PCR product, sometimes referred to as an ' ____________, ' is then in sufficient quantity that it can be easily measured by a variety of techniques.

30

This molecular ' Xeroxing ' process involves heating and cooling samples in a precise thermal cycling pattern over ~_____ cycles.

genotyped

To aid discovery of laboratory contamination, everyone in a forensic DNA laboratory is typically _______________ in order to have a record of possible contaminating DNA profiles. This is often referred to as a staff elimination database.

A common solution to overcoming or reducing PCR inhibition is:

To dilute the genomic DNA template, which also dilutes the effective concentration of the PCR inhibitor.

The purpose of a positive control is:

To ensure confidence that the reaction components and thermal cycling parameters are working for amplifying a specific region of DNA.

biochemical

To scale down _____________ reactions and provide capabilities for single-cell analysis, a German company named Advalytix (Munich) has developed an on-chip PCR plat- form involving a microscope slide containing 48 hydrophilic spots (although earlier versions had 60 spots).

35

Using different DNA polymerases and a faster temperature ramp rate, PCR amplifications of STR typing kits have been reduced to approximately _______ minutes on a conventional GeneAmp 9700 thermal cycler, which can change temperatures at a maximum rate of 4 ° C/second.

protocol

Various thermal cycling parameters including annealing temperatures and extension times are often examined in developing the final _____________.

hexamers

WGA amplifies the entire genome using random ____________ as priming points.

important

Well -designed primers are probably the most _____________ components of a good PCR reaction.

stochastic fluctuation

When amplifying very low levels of DNA template, a phenomenon known as ___________ ______________ can occur.

variation

When performing a common test on a number of different samples, the goal should be to examine the ____________ in the DNA samples, not variability in the reaction components used and the sample preparation method.

nanogram

While it is possible that WGA may play a limited role in enriching samples for archiving purposes that are in the low ____________ range, it will probably not be the end-all solution to low copy number DNA samples.

genome

Whole ___________ amplification prior to PCR and locus-specific amplification also have the same issues in terms of stochastic fluctuations with low levels of DNA.

15

With faster ramp rate cyclers, STR typing results have been obtained in as little as _______ minutes.

availability

With the widespread _______________ of commercial kits, individual forensic laboratories rarely perform PCR optimization experiments any more.

copies

Without the ability to make _________ of DNA molecules, many forensic samples would be impossible to analyze.

molecular crowding

Work with ' ___________ ___________ ' materials such as polyethylene glycol, where the amount of DNA is enriched in localized areas of a sample, has shown improved success with STR typing from low amounts of DNA.

1

Yields of 4 - 7 μ g of amplified genomic DNA are possible from as little as ____ ng of starting material.

Because the polymerase is busy amplifying these competing products, the target DNA region will be amplified less efficiently. What results then?

You will get less of what you are looking for and you may not have enough specific DNA to run your other tests.

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.

BSA

_________ is a common ingredient in most STR typing kits.

outdoor

__________ crimes may leave body fluid such as blood and semen on soil, sand, wood, or leaf litter that contain substances which may coextract with the perpetrator's DNA and prevent PCR amplification.

WGA

__________ involves a different DNA polymerase than the TaqGold enzyme commonly used in forensic DNA analysis.

controls

___________ are used to monitor the effectiveness of the chosen experimental conditions and/or the technique of the experimenter.

thermal

___________ stable polymerases that do not fall apart during the near boiling denaturation temperature steps have been important to the success of PCR.

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.

extension

_______________ times during thermal cycling are often increased for multiplex reactions in order to give the polymerase time to fully copy all of the DNA targets.