A Project-work on

Methods and Costs of

DNA Fingerprinting

Life Science III

Plant and Animal Biotechnology

(Towards partial fulfilment of the evaluation of the subject)

SUBMITTED BY: SUBMITTED TO:

KARAN SINGH and ANIRUDH SINGH DR. ANJANA VYAS

Roll No. – 977 and 973 FACULTY OF LAW

Sem. – VI, Sec. – ‘B’ NLU, JODHPUR

NATIONAL LAW UNIVERSITY JODHPUR

4th

February, 2015

ACKNOWLEDGEMENT

We owe our heartiest thanks to our subject teacher Dr. Anjana Vyas for showing

confidence in us and for giving us the opportunity to learn and understand the different

aspects of the topic “Methods and Costs of DNA Fingerprinrting”. We pay our sincere

regards to our subject teacher for encouraging us at each and every step whenever it was

required.

We would like to thank the Library Staff at National Law University, Jodhpur, for

having been extremely co-operative and ever willing to lend a helping hand to us during the

course of our research and analysis for the purpose of this term paper.

It is indeed a great pleasure and privilege to express our special thanks to colleagues

and other well-wishers who in different ways have given us splendid help, valuable

suggestions and encouragement. We thank almighty God for his blessings and giving us

strength to complete the project successfully.

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TABLE OF CONTENTS

Acknowledgement…………...…………………………………………….…….i

Introduction……………………………………………………………………...1

Methods of DNA Fingerprinting………………………………………………..2

Polymerase Chain Reaction (PCR)……………………………………………..3

Restriction Fragment Length Polymorphism (RFLP)…………...……………...9

Amplified Fragment Length Polymorphism (AmpFLP)...…………………….11

Short Tandem Repeats (STR)……………………………………………….…16

INTRODUCTION

DNA fingerprinting, one of the great discoveries of the late 20th century, has

revolutionized forensic investigations. This review briefly recapitulates 30 years of progress

in forensic DNA analysis which helps to convict criminals, exonerate the wrongly accused,

and identify victims of crime, disasters, and war.

DNA fingerprinting is one of the greatest identification systems we have to recognize

an individual or living organism. Every living creature is genetically different in its own way,

except for identical twins, triplets etc. DNA is comparable to a serial number for living

things. Each individual contains a unique sequence that is specific to that one organism.

Unlike traditional fingerprints which can be surgically altered or self-mutilated, the DNA

sequence cannot easily be changed once the material is left at a crime scene, thus increasing

its effective use in forensics, and the probability of finding an exact match. This method of

identification is useful in many applications such as forensics, paternity testing, and

molecular archaeology.

Up through 1984, the only method of establishing and authenticating personal

identification was by the fingerprint process. Since no two humans have been found to have

identical pattern of ridges on their fingers, this method has been universally accepted as a

means of personal identification. In 1984, Sir Alec Jeffreys was able to distinguish

differences among individuals based solely on their DNA composition. Since this

advancement in forensic science was announced in 1985, there has been tremendous progress

made in the methodology of extracting the DNA samples from such things as blood, saliva,

personal items and in the identification of human remains.

The purpose of this chapter is to provide a basic understanding of the molecular

protocols used for DNA fingerprinting or DNA profiling.

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METHODS OF DNA FINGERPRINTING

DNA (deoxyribonucleic acid) represents the blueprint of the human genetic makeup.

It exists in virtually every cell of the human body and differs in its sequence of nucleotides

(molecules that make up DNA, also abbreviated by letters, A, T, G, C; or, adenine, thymine,

guanine, and cytosine, respectively). The human genome is made up of 3 billion nucleotides,

which are 99.9% identical from one person to the next. The 0.1% variation, therefore, can be

used to distinguish one individual from another. It is this difference that can be used by

forensic scientists to match specimens of blood, tissue, or hair follicles to an individual with a

high level of certainty.

The complete DNA of each individual is unique, with the exception of identical twins.

A DNA fingerprint, therefore, is a DNA pattern that has a unique sequence such that it can be

distinguished from the DNA patterns of other individuals. DNA fingerprinting is also called

DNA typing.

There have been a number of techniques developed over the years, but this chapter

will focus on the more current (or generally considered to be more consistently informative)

techniques, namely the polymerase chain reaction (PCR), DNA sequencing, amplified

fragment length polymorphism (AFLP), and microsatellite analysis of short tandem repeats

(STRs). It will be assumed that the reader possesses a basic knowledge of the structure and

chemical properties of DNA.

Although many different DNA fingerprinting systems are available, the ones

discussed in this chapter are those most commonly used in the forensic individualization of

biological evidence, both from human and nonhuman sources.

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POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction, better known as PCR, is one of the technologies that not

only made a tremendous impact on the scientific community, but also affected many aspects

of our everyday lives. More than 30 years ago, the introduction of recombinant DNA

technology as a tool for the biological sciences revolutionized the study of life. Molecular

cloning allowed the study of individual genes of living organisms; however this technique

was dependent on obtaining a relatively large quantity of pure DNA. This depended on the

replication of the DNA of plasmids or other vectors during cell division of microorganisms.

Researchers found it extremely laborious and difficult to obtain a specific DNA in quantity

from the mass of genes present in a biological sample. A technique that amplifies DNA

through a simple enzymatic reaction was developed by Karry Mullis at that time which

enabled scientists to make millions - or even billions - of copies of a DNA molecule in a very

short time.

PCR has transformed the way that almost all studies requiring the manipulation of

DNA fragments may be performed as a result of its simplicity and usefulness. In 1993, Mullis

got Nobel Prize in Chemistry for his dedicated work on PCR. It has been used to detect DNA

sequences, to diagnose genetic diseases, to carry out DNA fingerprinting, to detect bacteria or

viruses (particularly the AIDS virus), and to research human evolution. Previous techniques

for isolating a specific piece of DNA relied on gene cloning - a tedious and slow procedure.

PCR, on the other hand we pick the piece of DNA we're interested in and have as much of it

as we want. Within a few years PCR - took the world's biological laboratories by storm. The

polymerase chain reaction reaped the highest scientific honour for its inventor in record time

as it provided a solution to one of the most pressing problems facing biology at the time - the

replication of DNA.

PCR is a rapid, inexpensive and simple way of copying specific DNA fragments from

minute quantities of source DNA material, even when that source DNA is of relatively poor

quality. It does not necessarily require the use of radioisotopes or toxic chemicals. There are

two reasons why you may want to amplify DNA.

1. You may want to simply create multiple copies of a portion of DNA which is very

rare. For example a forensic scientist may needs to amplify a small fragment of DNA

from a crime scene.

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2. You may wish to compare two different samples of DNA to know which is the more

abundant. Because DNA is microscopic so you cannot see which sample contains the

most DNA. However, if you amplify both samples at the same rate, you can calculate

which sample was the biggest to begin with by establishing which is the biggest after

amplification.

It is the Polymerase enzyme that drives a PCR. A polymerase will synthesize a

complementary sequence of bases to any single strand of DNA providing it has a double

stranded starting point. This is very useful because you can choose which gene you wish the

polymerase to amplify in a mixed DNA sample by adding small pieces of DNA

complimentary to your gene of interest. These small pieces of DNA are known as primers

because they prime the DNA sample ready for the polymerase to bind and begin copying the

gene of interest. During a PCR, changes in temperature are used to control the activity of the

polymerase and the binding of primers.

To begin the reaction the temperature is raised to 95oC. At this temperature all double

stranded DNA is "melted" in to single strands:

The temperature is then lowered to ~50oC. This allows the primers to bind to your

gene of interest. Thus the polymerase has somewhere to bind and can begin copying the DNA

strand:

The optimal temperature for the polymerase to operate is 72oC so at this point the

temperature is sometimes raised to 72oC to allow the enzyme to work faster. There are now

twice as many copies of your gene of interest as when you started:

The cycle of changing temperatures (95oC, 50

oC and 72

oC) is then repeated and two

copies become four. Another cycle and four become eight, up to 30-35 cycles. After

amplifying your gene into many millions of copies it is possible to run the amplified DNA

out on an agarose gel and stain it with a dye to visualize it. The bigger the visible band,

contains more copies of gene of interest that you have created.

WORKING PRINCIPLE OF PCR:

As the name implies, it is a chain reaction, a small fragment of the DNA section of

interest needs to be identified which serves as the template for producing the primers that

initiate the reaction. One DNA molecule is used to produce two copies, then four, then eight

and so forth. This continuous doubling is accomplished by specific proteins known as

polymerases, enzymes that are able to string together individual DNA building blocks to form

long molecular strands. To do their job polymerases require a supply of DNA building

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blocks, i.e., the nucleotides consisting of the four bases adenine (A), thymine (T), cytosine

(C) and guanine (G). They also need a small fragment of DNA, known as the primer, to

which they attach the building blocks as well as a longer DNA molecule to serve as a

template for constructing the new strand. If these three ingredients are supplied, the enzymes

will construct exact copies of the templates.

Figure 1: Number of copies of DNA obtained after 'n' cycles = 2(n+1)

It is then possible to clone DNA whose sequence is unknown. This is one of the

method's major advantages. Genes are commonly flanked by similar stretches of nucleic acid.

Once identified, these patterns can be used to clone unknown genes - a method that has

supplanted the technique of molecular cloning in which DNA fragments are tediously copied

in bacteria or other host organisms. With the PCR method this goal can be achieved faster,

more easily and above all in vitro, i.e., in the test-tube. Moreover, known sections of long

DNA molecules, e.g. of chromosomes, can be used in PCR to scout further into unknown

areas.

PARAMETERS THAT AFFECT PCR:

Essential components of polymerase chain reactions:

1. A thermostable DNA polymerase to catalyse template-dependent synthesis of

DNA:

Depending on the ability, fidelity, efficiency to synthesize large DNA products, a

wide choice of enzymes is now available. For routine PCRs, Taq polymerase(0.5-2.5

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units per standard 25-50 uL reaction)remains the enzyme of choice. The specific

activity of most commercial preparations of Taq is ~80,000 units/mg of protein. Since

the efficiency of primer extension with Taq polymerase is generally ~0.7, the enzyme

becomes limiting when 1.4*1012 to 7*1012 molecules of amplified product have

accumulated in the reaction.

2. A pair of synthetic oligonucleotides to prime DNA synthesis:

Design of the oligonucleotide primer being the most important factor that influence

the efficiency and specificity of the amplification reaction, careful designing of

primers is required to obtain the desired products in high yield, to suppress

amplification of unwanted sequences and to facilitate subsequent manipulation of the

amplified product. Since the primers so heavily influence the success or failure of

PCR protocols, it is ironic that the guidelines for their design are largely qualitative

and are based more on common sense than on well understood thermodynamic or

structural principles.

Design of oligonucleotide primers for PCR

In certain situations, it may be desirable to amplify several segments of target DNA

simultaneously. In these cases, an amplification reaction named "multiplex PCR" is

used that includes more than one pair of primer in the reaction mix. Standard

reactions contain non-limiting amount of primers, typically 0.1-0.5μM of each primer

(6*1012 to 3*1013 molecules). This quantity is enough for at least 30 cycles of

amplification of a 1 kb segment of DNA. Higher concentrations of primers favour

mispriming which may lead to nonspecific amplification.

3. Deoxynucleoside triphosphates(dNTPs)

Standard PCRs contain equimolar amounts of all four dNTPs Concentrations of 200-

250μM of each dNTP recommended for Taq polymerase in reactions containing 1.5

mM MgCl2. In a 50 uL reaction, these amounts should allow synthesis of ~6-6.5μg

DNA which should be sufficient even for multiplex reaction in which eight or more

primer pairs are used at the same time. High concentrations of dNTPs (>4mM) are

inhibitory, perhaps because of sequestering of Mg2+. However, a satisfactory amount

of amplified product can be produced with dNTP concentrations as low as 20μM- 0.5-

1.0pM of an amplified fragment ~1 kb in length. To avoid problems, stocks of dNTPs

7

should be stored at -20oC in small aliquots that should be discarded after the second

cycle of freezing or thawing. During long term storage at -20oC, small amounts of

water evaporate and then freeze on the walls of the vial. To minimize changes in

concentration, vials containing dNTP solutions should be centrifuged, after thawing,

for a few seconds in a micro centrifuge.

4. Divalent cations

All thermostable DNA polymerases require free divalent cations- usually Mg2+ for

activity. Some polymerases will also work, albeit less efficiently with buffers

containing Mn2+. Calcium ions are quite ineffective. Because dNTPs and

oligonucleotides bind Mg2+, the molar concentration of the cation must exceed the

molar concentration of phosphate groups contributed by dNTPs and primers. It is

therefore impossible to recommend a concentration of Mg2+ that is optional in all

circumstances. Although a concentration of 1.5 mM of Mg2+ is routinely used,

increasing the concentration of Mg2+ to 4.5 mM or 6mM has been reported to

decrease nonspecific priming in some cases and to increase it in others. The optimal

concentration of Mg2+ must therefore be determined empirically for each

combination of primers and template. The preparations of template DNA should not

contain significant amount of chelating agents (like EDTA or negatively charged ions

like PO43-), which can sequester Mg2+.

5. Buffer to maintain pH

Tris–Cl, adjusted to a pH between 8.3 and 8.8 at room temperature is included in

standard PCRs at a concentration of 10mM. When incubated at 72oC (extension phase

of PCR), the pH of the reaction mixture drops by more than a full unit, producing a

buffer whose pH is ~7.2.

6. Monovalent cations

Standard PCR buffer contains 50mM KCl and works well for amplification of

segments of DNA >500bp in length. Raising the KCl concentration to ~70-100mM

often improves the yield of shorter DNA segments.

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7. Template DNA:

Template DNA containing target sequences can be added to PCR in single or double

stranded form. Closed circular DNA templates are amplified slightly less efficiently

than linear DNAs. All though the size of the template DNA is not critical,

amplification of sequences embedded in high molecular weight DNA (>10kb) can be

improved by digesting the template with a restriction enzyme that doesn't cleave

within the target sequence.

When working at its best, PCR requires only a single copy of target sequence as

template. More typically, however, several thousand copies of the target DNA are

seeded into the reaction. In the case of mammalian genomic DNA, up to 1μg of DNA

is utilized per reaction, an amount that contains ~3*105 copies of a single-copy

autosomal gene. The typical amounts of yeast, bacterial and plasmid DNAs used per

reaction are 10μg, 1μg and 1pg respectively.

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RESTRICTION FRAGMENT LENGTH POLYMORPHISM

Now that we have a better understanding of what DNA actually is, le us move on to the

basics of making a DNA fingerprint. There are three types of DNA fingerprints: RFLPs,

VNTRs, and STRs. Restriction fragment length polymorphisms, or RFLPs as they are

commonly known, were the first type of DNA fingerprinting which came onto the scene in

the mid- 1980’s. RFLP’s focus on the size differences of certain genetic locations. The first

step in creating an RFLP fingerprint is obtaining and isolating the DNA. DNA can be

obtained from almost any of the cells or tissues in the human body. You do not need a large

amount of tissue or blood to provide enough DNA for analysis. The DNA is then extracted

from the blood or tissue sample, and from here we carry out our second step in the process

which is the cutting, sizing, and sorting of the DNA sample. DNA is cut using restriction

enzymes, which cut the DNA stand at specific places. Restriction enzymes are usually

isolated from bacteria that use them to degrade foreign DNA like viral DNA. Each type of

restriction enzyme recognizes and cuts a particular DNA sequence.

The DNA at this point is cut into a various array of pieces which are sorted according by

size through a process called electrophoresis. In this process the DNA particles are mixed

into a buffer solution and applied to a gel made from seaweed agarose. Each side of the gel is

connected to an electrical current. The DNA is negatively charged due to its phosphate

groups, so it migrates towards the positive electrode or anode. The smaller pieces of DNA

move faster (sieve) through the gel than the larger ones, so this provides the basis of the

fragment separation. “This technique is the DNA equivalent of screening sand through a

progressively finer mesh screens to determine particle sizes”.

The band pattern that the DNA creates in the agarose gel is then transferred to a nylon

sheet. To complete this transfer a nylon sheet is placed on the gel and left to soak overnight in

a high salt solution. After the soaking procedure is completed, the nylon membrane contains

the same pattern of DNA as occurred in the original gel. The membrane is now prepared to

undergo its probing phase. Radioactive or fluorescently-8 labeled probes are hybridized onto

the nylon membrane, which bind to specific DNA sequences present in the pattern to produce

a pattern of bands which create the DNA fingerprint. This process can be performed with

several different probes simultaneously to make the final product which looks very similar to

the bar codes you see in retail stores. Figure 2 shows an actual RFLP-type DNA fingerprint.

Variable number tandem repeats, or VNTRs represent specific locations on a

chromosome in which tandem repeats of 9-80 or more bases repeat a different number of

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Figure 2: An example of a RFLP autoradiograph (RFLP Autorad Image, 2003).

times between individuals. These regions of DNA are readily analyzed using the RFLP

approach and a probe specific to a VNTR locus. The fragments are a little shorter than RFLPs

(about 1-2 kilo base pairs), but are created through the exact same process. Figure 3 shows an

example of a VNTR fingerprint.

Figure 3: An example of a VNTR autoradiograph

Since RFLPs and VNTRs are created in the same fashion, they exhibit the same overall

advantages and disadvantages. Some of the advantages of these types of DNA fingerprints

are that they are the most stable and reproducible, which is a valuable trait to have when you

are trying to determine an exact match of a person’s DNA, which must exclude billions of

other people’s DNA with a certain degree of confidence. They are also easier to prevent

contamination since the DNA sample is larger than with other types of DNA fingerprints, and

small amounts of DNA contamination does not alter the analysis. Some of the disadvantages

of RFLPs and VNTRs include they are very time consuming (especially the probe

hybridization step), relatively large amounts of DNA must be used to obtain an adequate

sample, too many polymorphisms may be present for a short probe, and the cost is very high

due to labor and time requirements.

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AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)

Amplified Length Fragments Polymorphism is a recent DNA fingerprinting technique

developed by Zabeau and Vos (1993); but see also Vos et al. (1995) and Vos and Kuiper,

1997). This method is based on PCR amplification of selected restriction fragments of a total

digested genomic DNA. Once labelled, amplified products are separated by electrophoresis.

DNA fragments obtained range from 60 to 500 base pairs.

To be visualised, DNA polymorphism, which is usually made of small DNA fragments of

few base pairs (up to 500), must be amplified. This amplification is commonly done by

Polymerase Chain Reaction (Mullis et al., 1986; Mullis and Faloona, 1987). The PCR method

can amplify specific DNA fragments through a precise priming of the polymerisation reaction

occurring at each end of the target DNA. This precise priming is done by short

oligonucleotidic sequences (Primers) able to anneal to the template DNA in the target zone.

Primers are 18-24 base pairs long, synthesised in laboratory and correspond to a

complementary DNA sequence designed in the flanking regions of the heavy strand of the

target DNA. The Polymerase Chain Reaction starts first with a high temperature phase

(denaturation) that produces single-stranded DNA. Then, once temperature has reached the

TM, primers will bind to the template DNA. The Taq polymerase recognises each double-

stranded DNA as a start of synthesis and will continue the polymerisation reaction in the

direction 5’ 3’ as soon as the temperature hasreached 72°C (optimal elongation

temperature).

Therefore, in order to design specific primers, the sequences of the flanking regions of

the target DNA must be known. This supposes detailed knowledge about the genome or

further elaborated investigations to get it. This step usually requires high laboratory

equipment and are, most of the time, time consuming.

The originality of the AFLP method was to design and synthesize arbitrary primers

first, and then to ligate them to target DNA fragments (Box 3). The AFLP arbitrary primers

are called “adapters” and consist of a known sequence of 20 nucleotides. The target DNA

sequences are DNA fragments generated by restriction enzymes. Fragments are produced

from total genomic DNA by the combined action of two restriction enzymes. Then, adapters

are ligated at each end of a restriction fragment by a protein ligase. Finally, adapters are used

in a PCR as priming sites to amplify the restriction fragments. AFLP markers reveal a

“restriction site” polymorphism and must be treated as dominant markers, since homozygotes

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and heterozygotes cannot be established unless breeding/pedigree studies are carried out to

determine inheritance patterns of each fragment. However, the large number of fragments

gives an estimate of variation across the entire genome, which thus gives a good general

picture of the level of genetic variation of the studied organism.

BASIC STEPS OF AFLP FINGERPRINTING:

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1. DNA Extraction

Clean and high molecular weight DNA is a prerequisite for AFLP. In our study, we

extracted DNA according to Doyle & Doyle method (Doyle and Doyle, 1988). This method

is based on the CTAB procedure. For more details, refer to the protocol (3.3) and

troubleshooting (3.4) parts.

2. Restriction

Restriction fragments of the genomic DNA are produced by using two different

restriction enzymes: a frequent cutter (the four-base restriction enzyme MseI) and a rare

cutter (the six-base restriction enzyme EcoRI) (Box 3). The frequent cutter serves to generate

small fragments, which amplify well and which have the optimal size range for separation on

a sequence gel, whereas the rare cutter limits the number of fragments to be amplified.

3. Ligation of Oligonucleotide Adapters

Double-stranded adapters consist of a core sequence and an enzyme-specific sequence

(Box 3). Therefore, adapters are specific for either the EcoRI site or the MseI site. Usually

restriction and ligation take place in a single reaction. Ligation of the adapter to the restricted

DNA alters the restriction site in order to prevent a second restriction from taking place after

ligation has occurred. The core sequence of the adapters consists of a known DNA sequence

of 20 nucleotides, which will be used later as primer in the PCR.

4. Pre-Amplification

This step is a normal PCR where the adapters are used as primers. This first PCR, called

pre-amplification, allows a first selection of fragments by only amplifying the DNA

restriction fragments that have ligated an adapter to both extremities. Additionally to the

adapter sequences, the primers used for the pre-selective amplification have a supplementary

base. This extra base enables another first selection by amplifying ¼ of the fragments that

have ligated an adapter to both extremities. These first three steps (DNA extraction,

restriction/ligation and pre-amplification) can be run and visualised on a 1.6 % agarose gel

(Figure 4).

5. Amplification

The aim of this step is to restrict the level of polymorphism and to label the DNA. For

this second amplification, we added three more nucleotides at the 3’ end of the primer

sequence used for the pre-amplification (= adapters sequence + 3 nucleotides; Box 3). These

two additional nucleotides make the amplification more selective and will decrease the

number of restriction fragments amplified (polymorphism). Moreover, one of the primers

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(usually the EcoRI primer) is labelled with a fluorescent dye, and will allow the visualisation

of DNA during the migration.

Figure 4: The three first steps of the AFLP process (DNA extraction, restriction/ligation and pre-amplification)

run on a 1,6% agarose gel. The 10 first lanes (A) represent genomic DNA of 10 Cirsium arvense samples.

According to the Lambda DNA concentration standards (B from left to right: 0.125; 0.25; 0.5; 0.75; 1; 1.5; 2

µg) C. arvense DNA concentration can be estimated to be in around 5ng per lane (5µl DNA load). (C)

restriction/ligation of the same 10 samples. Genomic DNA was restricted with Mse I and EcoR I enzymes. The

restriction produces a large quantity of small size DNA fragments. During the pre-amplification (D), only a part

of the restricted fragments are amplified. The pre-amplification leads to a homogeneous DNA smear ranging

from 100 to 800 bp. (λ) represents a DNA size marker.

6. Electrophoresis

The PCR products are denaturated and run on acrylamide gel (DNA sequencer). In our

study, samples were run on an ABI Prism 310 (Figure 5). A thin capillary containing a

polymer replaces the usual acrylamide gel. The electrophoresis conditions we used for

fragments analysis can resolve DNA fragments differing just by one base pair. Samples are

loaded in a track, and run one after the other through the capillary.

All fragments are separated with regard to length, smaller fragments running first.

Once passing the laser, a dye attached to the primer is excited and emits a fluorescent signal

15

that is then collected by a computer. The results of fluorescence are visualised on the

computer as peaks, called Electropherograms (Figure 6). Each peak corresponds to a band on

a normal acrylamide gel. Amplified fragments range from 30 to 400 base pairs.

Figure 5: ABI Prism Sequencer

Figure 6: AFLP electropherogram. On the figure, each peak corresponds to a DNA restriction fragment of a

precise length. From the left to the right, the 5 shaded peaks correspond to DNA fragments having respectively

131; 161; 190; 198 and 242 base pairs.

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SHORT TANDEM REPEATS

The tandemly repeated DNA units of mini- and microsatellite loci are often very useful

for genotyping due to their typically high level of polymorphic variation in a population. This

last section will discuss what these loci are and what must be taken into consideration to

properly amplify and interpret the results when using them for genotyping. Microsatellite

sequences, now more commonly referred to as short tandem repeats (STRs), have a repetitive

unit of two to six bases in length, repeated in a tandem or head-to-tail orientation (see Figure

6). The satellite nomenclature comes from early studies in which genomic DNA was isolated

and then fractionated using density gradients. Fractions were analyzed with

spectrophotometry and then each fraction’s density was plotted against their absorbency

values. It was found that the bulk of the genomic DNA was collected in one fraction and

produced the main absorbance peak, but there were also one or more secondary, or satellite,

absorbance peaks. These fractions were found to contain AT-rich repetitive DNA sequences

typically associated with the centromere or telomere regions of chromosomes. Satellite DNA

soon came to mean any tandemly repeated DNA. The mini and micro prefixes were used for

repetitive DNAs that were composed of shorter repeat units with a lower copy number of this

unit. Figure 6A provides an example of two possible alleles of a hypothetical locus with a

CAGT repeat. One allele has six copies of the repeat sequence while the other has nine

copies. Regions of conserved sequence just upstream and downstream of the STR locus are

used to design primers for PCR amplification of that site. Because any two alleles will

typically differ only in the copy number of the STR, the difference in length of each amplicon

will be whole multiples of the four-base repeat. For this reason, alleles are designated by the

number of tandem repeats they contain. The reason these loci are typically so polymorphic

and the alleles often differ in length by whole multiples of the repeat unit is due to strand

slippage or stutter during replication.

Experimental evidence supports the idea that when an extending DNA polymerase is

released prematurely, the incomplete DNA strand can denature from the template and then

reanneal. If this occurs in the region of the repeated units, the extended strand can anneal in a

displaced, or out-of-register, fashion due to the repetitive nature of the sequence. The fact that

the base complementarity is a short unit in a tandem organization, a small kink in either

strand allows for annealing of the last few bases of the new strand to a repeat unit preceding

or following the one it was first replicated from. If a new polymerase molecule begins

17

.

Figure 7: An example of an STR electropherogram with a commercially available allelic ladder (mixture of

DNA fragments of known size for comparison to test samples) and a positive amplification control used to

confirm that the STR kit is performing as expected.

from this displaced strand, a DNA duplex with strands of unequal length will be generated

with the length difference being some multiple of the repeated unit. A size difference of a

single repeat unit is the most common. In vivo, these unequal strands will be corrected by

DNA repair mechanisms usually back to the length of the original allele. Occasionally, the

DNA duplex can be repaired such that a new allele is generated. If this occurs during the

formation of a germ cell and this germ cell becomes part of a zygote, a new allele or mutation

is generated. During in vitro DNA replication (PCR), these unequal strands will be denatured

and used as templates in the next round of amplification and thus will result in a mixture of

PCR products. When analyzed on an acrylamide gel, there will not only be a peak

representing the true size of the allele, but one or more peaks representing PCR stutter

products that differ in size from the true peak by whole multiples of the repeat unit.

Experimental evidence has shown that longer repeat units are less susceptible to the

production of stutter amplicons. This is why STR loci of four bases or more are used for

forensic applications. Stutter can still occur for such loci (see Figure 7), but the amplification

of such products is typically fewer than 10% of the true allele as measured by peak height.

Levels of stutter higher than this would be a significant problem when trying to determine if a

18

sample of genomic DNA from an unknown source was from a single individual or a mixture

of different individuals (not an uncommon occurrence at a crime scene). If two individuals

were contributors to a DNA sample and the contribution of one individual was small in

comparison to the other, then the smaller peak heights of the allele amplicons of the minor

contributor could be mistaken for stutter products or vice versa. Thus far, we have discussed

lengths of STR alleles always being whole multiples of the repeated unit due to a mutational

mechanism caused by stutter. Obviously, other types of mutational events occur in genomic

DNA and can thus occur in an STR allele. Two such events are point mutations and insertion

or deletions of nucleotide bases. A point mutation is when a base pair is changed from one

form to another; for example, an A-T base pair mutating to a G-C. Insertion or deletion

mutations are exactly that, and can be of one or more base pairs. The impetus for such

mutations can be exposure to mutagenic agents or spontaneous due to chemical tautomeric

shifts of the nucleotides during replication. If such mutations occur in an STR allele, then

there will either not be a size change or the size change will most likely not be a whole

multiple of the repeated unit. Such STR allelic variants are known as microvariants. Figure

6B illustrates two possibilities for a deletion variant. The deletion is of a single base pair in

both examples. In the first example, the deletion is an A-T base pair from the seventh CAGT

repeat of the original allele, while the second is a C-G base pair in the region outside of the

repeated units, but still within the region amplified by the primers. Amplicons of both of

these alleles would be the same length and would be known as 8.3 alleles since they are one

base pair shorter than a 9 allele. The only way to determine that these 8.3 alleles are actually

different would be to sequence them. A number of such allele types have been recorded for

many STR loci in use today. Microvariants, while noted, do not interfere with the ability to

type an individual and in fact often lend an extra bit of uniqueness to a DNA STR profile.

Another type of amplicon, or peak, artifact that can occur in STR analysis is known as

nontemplate addition. The most commonly used thermostable DNA polymerases have the

propensity to add an extra A base to the 3′ strand ends of the PCR amplicons. When this

occurs, the denatured amplicon strands will obviously be one base longer than the length

spanned from one primer to the other. This is not a problem as long as this occurs to all of the

amplicon molecules. This would make every molecule one base longer and thus there would

be no relative change from one fragment to another. If both types are present in the

amplification, then a double peak, or a peak with a shoulder, will be produced in the gel

separation and analysis. Because the frequency with which this occurs can vary due to the

amplifica-tion conditions, amplification protocols are designed to produce 100% non-

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template addition so only a single amplicon size, or peak, is produced for each allele. This is

accomplished by putting enough nucleotides into the reaction so they are not a limiting

–45 minutes in duration at the end of the amplification

temperature cycling profile. This ensures that almost every amplicon molecule has an A base

added to both its 3′ strand ends.

Figure 7 is an electropherogram (i.e., software output) for a human commercial STR kit,

COfiler (Applied Biosystems, data courtesy of Craig O’Connor, University of Connecticut).

The kit contains reagents to amplify six STR loci (D3S1358, D16S539, THO1, TPOX,

CSF1P0, and D7S820) and one sex chromosome locus (Amelogenin) from human genomic

DNA. Amelogenin is not an STR but allows for sex determination of the contributor of an

unknown genomic sample. The gene exists on both the X and Y human chromosomes, but

the X version has a six-base-pair deletion relative to the Y version, allowing for size

separation during electrophoresis if both are present. Just as for AFLP, when separating STR

amplicons, an internal lane size standard (GeneScan 500ROX, Applied Biosystems Inc.) is

added to each sample lane to allow for adjustment of slight lane-to-lane differences during

electrophoresis. In addition, since virtually all the alleles present in human populations for

these loci are known, an allelic ladder is loaded into several lanes (along with the same

internal lane size standard). Using different flourophore tags for loci that have some allelic

amplicons within the same size range allows for more loci to be amplified in a single reaction

tube and analyzed in one lane of the gel. A direct comparison between lanes containing the

allelic ladder and those containing an unknown sample generates a DNA profile of the

individual for these seven loci. For the human sample shown in Figure 7, the individual

would be typed: female (lack of Y-allele sized amplicon); (14,15) D3S1358 heterozygote;

(11,12) D16S539 heterozygote; (8,9.3) THO1 heterozygote; (8,8) TPOX homozygote;

(10,12) CSF1P0 heterozygote; and (10,11) D7S820 heterozygote.

If enough previous data of genotypes of many individuals from many populations have

been collected, estimated allele frequencies within the populations can be calculated. Using

these estimated allele frequencies, an expected genotypic frequency can be calculated for

each locus. Where p and q represent allele frequencies, p2 or 2pq (homozygous or

heterozygous conditions, respectively) would be used to calculate the expected frequency of

that particular genotype for each locus. To generate an expected frequency for all seven loci

combined, one would take the product of the expected genotypic frequencies for each

individual locus. The main impetus for making such a calculation in forensics is for the

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benefit of a typical layperson that would be sitting on a jury. Any DNA expert would

recognize the full significance of a suspect sharing the same DNA profile as that left at a

crime scene, and that the probability of two individuals (except for identical twins) matching

at all seven loci is essentially zero. But obviously, given that it is a probability estimate, it is

still within the realm of possibility. In fact, most forensic laboratories report STR profiles for

a standardized set of 13 loci. Therefore, to be able to communicate the significance of a

suspect being included as a donor of a DNA sample, the expected frequency of that genotype

in the human population is calculated and reported as a random match probability.

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COST ANALYSIS OF DNA FINGERPRINTING

With DNA investigations becoming more and more common, we have to consider the

cost related to this type of investigation. What types of cases should it be used for and how

much is it costing us? A major disadvantage of DNA fingerprinting is the cost. Many local

police cannot afford a full time DNA fingerprint analyst. This leads them to outsource the

tests to experts in other areas.

It was not long ago when law enforcement agencies paid $8,000 to $10,000 per case

to have experts to testify for criminal cases in places like New York and California. Now

with DNA testing, crime evidence can be gathered from blood, furnishing, saliva and

other evidence in local labs. This change in how evidence can be provided has reduced the

cost in some crime cases. In fact, in Nebraska, DNA was used as evidence in approximately

80 criminal cases. The cost is $90,000. Comparing this cost to $10,000 for just one

case shows that DNA testing has a positive impact on cost when relating it to the number of

cases that can be investigated and solved in communities.

Over the past 15 years, DNA testing for criminal cases has grown

tremendously. With more than 150 public forensic laboratories across the states, thousands

of DNA tests are conducted annually. This type of activity for DNA testing has brought

increased revenue in the sciences and increased public and private services for individuals

and businesses. Budgeting impacts become an issue when there is a backlog of criminal

cases that require DNA testing. In fact, in Wisconsin, the average cost for DNA testing is

$390.00 per case. However, when the State Crime Laboratory received 2,226 DNA cases in

2006 and was only able to process 1,152 cases, that savings quickly became a

cost. They projected that millions of dollars would be needed for hiring and training DNA

specialist. So what with DNA appearing to be a cost saving, it could quickly become a cost

burden based on the number of backlog cases in a criminal laboratory.

DNA testing is a valuable service and can be cost effective. It has increased the

efficiency and the speed in which criminal cases can be solved. As DNA becomes more

popular, it is important the law enforcement agencies reduce backlog cases in order to reduce

long term costs.