“DNA FINGERPRINTING IN HUMAN HEALTH AND

SOCIETY ”

BY

RAVINDRA KUMAR PANCHAL

(08BT000122)

&

AGRAWAL PARTH TARAPRAKASH

(08BT000105)

GUIDED BY:

Dr. NAVNEET SINGH CHAUDHARY

Submitted in partial fulfillment of the Degree of Bachelor of Technology

SESSION 2010-2011

DEPARTMENT OF BIO TECNOLOGYSIR PADAMPA T SINGHANIA UNIVERSITY UDAIPUR

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ACKNOWLEDGMENT

First and foremost, we would like to express my sincere gratitude to my report guide, Dr.

Navneet Singh Chaudhary . We were privileged to experience a sustained enthusiastic and

involved interest from his side. This fuelled our enthusiasm even further and encouraged us

to boldly step into what was a totally dark and unexplored expanse before us.

We would also like to thank our seniors who were ready with a posi-tive comment all the

time, whether it was an off-hand comment to encour-age us or a constructive piece of

criticism.

We would like to thank the SPSU staff members and the institute, in general, for extending a

helping hand at every juncture of need. We would like to express our gratitude towards our

parents for their kind co-operation and encouragement which help us in completion of this

project.

Last but not least, our thanks and appreciations also go to our col-leagues in developing the

project and people who have willingly helped us out with their abilities.

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CONTENTS 1. Introduction………………………………………………………… 4-101.1 Abstract 41.2 Executive summary 4-51.3 Report objective 5-6 1.4 dna(deoxyribonucleicacid) 7 1.5 about dna fingerprinting 81.6 what is dna fingerprinting 9 1.7 principles of dna fingerprinting10 1.7.1 base pairing102. methods of dna fingerprinting ……………………….…………….11-342.1 RFLP 12-27

2.1.1 steps of RFLP 14-26 Isolation of DNA.15

Cutting, sizing, and sorting. 15-22

Transfer of DNA to nylon.22-24

Probing.25

2.1.2 analysis of dna fingerprints26-27 2.2 PCR 2.3 AmpFLP2.4.STR 3. applications of dna fingerprinting………………………………....35-43 3.1 dna forensic.3.2 Paternity Testing.3.3 MIA Soldiers. 3.4 Inherited Disorder Testing. 3.5 Personal Identification.3.6 DNA Forensics Databases .

4. Conclusion……………………………………………………………….445. References………………………………………………………………………………………….45

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CHAPTER 1:INTRODUCTION

1.1 ABSTRACT

This project investigated the impact of the new technology of DNA fingerprinting on society,

especially the legal system and database ethics. Our conclusions propose an expansion of

DNA databases to include individuals convicted of any felony, not just violent crimes. The

benefit to society of such DNA databases to identify unknown corpses, determine paternity,

place a suspect at the scene of a crime, develop leads where otherwise there were none, and

link crimes to identify serial criminals outweighs any privacy issues especially for convicted

felons.

1.2 EXECUTIVE SUMMARY

Five decades ago, Watson and Crick discovered the secrets of DNA structure. DNA

Fingerprinting, or DNA profiling, was first adopted in 1984 by Oxford University educated

Alec Jeffreys. Jeffrey's discovery opened a whole new world that, once proven and perfected,

would unlock markers to visualize each person's unique identity. Inside each human being, as

well as plants, animals, and microorganisms, lies a unique DNA structure. Today, DNA

Fingerprinting is "rapidly becoming the primary method for identifying and distinguishing

individual human beings” (Betsch, 1994). Some of the applications of DNA fingerprinting

techniques include: murder cases, rape cases, paternity testing, diagnosis of inherited

disorders, military identification, and molecular archaeology. Forensic DNA analysis has

been admitted into United States courtrooms since 1987. DNA profiling does not claim to be

an absolute identification, but may be very strong evidence and is considered to be just one

part of the entire case. DNA profiling is used primarily in sexual assault cases. Prior to DNA

testing, labs were limited regarding the amount of genetic information that could be obtained

from evidence samples. Now with DNA testing, the genetic content of the evidence sample

itself can be examined. In the case of a rape investigation, four profiles are formed: the first is

a DNA profile from the blood of the victim, the second DNA profile is formed from the

blood of the defendant, thirdly a vaginal swab is taken, and the female and male fractions are

separated and profiled separately. The victim’s blood profile and the vaginal swab profile

should match alleles. The defendant’s blood sample profile should match the male fraction

profile in order to consider him as a suspect. It is important to remember that DNA profiling

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does not incriminate a suspect with absolute certainty but if the profiles don’t 5 match it can

exclude with absolute certainty. In the case of likely matches, statistics are used to determine

the frequency that this DNA profile likely appears in the general human population

.

"Since the discovery of DNA fingerprinting at the turn of the 20th century, science has

assumed an increasingly important and powerful role in the decision making process of our

judicial branch" (Biancamano, 1996). Many Landmark cases, which set the standards for

admitting DNA into the courtroom, as well as the reliability and acceptability of DNA

techniques, will be discussed to prove the methods used today are valid and reliable when

performed properly. Some of the earlier cases that will be talked about did not actually

involve DNA, but what they did accomplish was to set precedence by establishing new rules

for admittance of technical evidence, and to set new generally accepted scientific standards in

the court of law. Some of these landmark cases include: Frye v. United States in 1923,

Federal Rules of Evidence 702 (Rule 702) in 1975, Colin Pitchfork in 1986, Andrews v

Florida in 1988, People v Castro in 1989, Two Bulls v US in 1990, and the case of Paul

Eugene Robinson in 2003 where the DNA evidence was the primary basis of the conviction

of sexual assault.

1.3 REPORT OBJECTIVE:

DNA fingerprinting is a powerful new technology, which is used to assist in convicting

the guilty and exonerating the innocent. The topic of DNA fingerprinting however remains

controversial in the courtroom regarding technical issues, and also has legal, cultural and

political consequences. This controversy is mainly derived from the lack of knowledge

regarding the procedures of DNA fingerprinting, how it is obtained, analyzed, and reported.

The purpose of this project is to help to eliminate the public’s doubt, concerning DNA

fingerprinting and help them to convert their reservations about the science into support for

the use of DNA fingerprinting in the courtroom. The focus of this paper will be directed

toward the layperson in order to target the prospective population of jurors. Jurors are

members of our community who are required to evaluate the relevance of all evidence

including DNA evidence. Thus, once they are on a jury, and DNA evidence is brought in,

they will be able to comprehend the data and make an informed verdict. This paper will cover

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the primary areas of the public’s concerns, as well as provide history of DNA analysis, and

future advances of the science for admission into the courtroom. Description of the lab report,

as well as the actual data used to formulate the lab report will help the public develop an

appreciation for the formulation of the results of the actual testing. The objective of this paper

was accomplished by first, outlining the science of the main techniques of DNA

fingerprinting in laymen’s terms. Secondly the project describes the processes of collection,

storage, and prevention of contamination of the DNA evidence. Thirdly, landmark DNA

court cases that established legal precedents for admitting DNA in U.S. courts will be

revisited and analyzed. Lastly, many of the controversies regarding the DNA databases will

be analyzed. This project not only informs the reader about the facts of the history of DNA 8

fingerprinting, but it will also entice the reader to encourage and support the use of DNA

evidence in the courtroom.The overall goal of this paper is for the reader to walk away with

an understanding of DNA fingerprinting’s positive impact on society.

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1.4 DNA(Deoxyribonucleic acid):

Deoxyribonucleic acid, more commonly known as DNA, is the complex chemical structure

that uniquely identifies each and every organism. An organism’s complete set of DNA is

known as a genome. DNA is the fundamental building block of the genome (An Introduction

to DNA, 2002). DNA is located inside an organism's chromosomes.

"DNA itself is a double-helix polymer. A polymer is simply a large molecule that is

produced by several smaller units linking together and forming a chain" (Rohloff, 2000).

Figure – Watson and Crick’sdiscovery of the DNA “DoubleHelix” (Devitt, 2002)

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1.5 ABOUT DNA FINGERPRINTING

DNA Fingerprinting, or DNA profiling, was first adopted in 1984 by Oxford University

educated Alec Jeffreys. Jeffreys stumbled upon this theory while working on myoglobin, the

gene that codes for an iron-containing protein in muscles.

Jeffrey's discovery opened a whole new world that, once proven and perfected, would unlock

each person's unique identity. Inside each and every human being, as well as plants, animals,

and microorganisms, lies a unique DNA structure .Conventional fingerprints occur only on

the fingertips, and are capable of being altered, but a DNA fingerprint is the same for"every

cell, tissue, and organ of a person”. DNA is incapable of being altered, and is "rapidly

becoming the primary method for identifying and distinguishing among individual human

beings”. In science, DNA Fingerprinting does not point to a unique individual. However, it

provides a profile, and then the probability that there are others who also match the profile is

determined leading to the match or conviction. So what is DNA fingerprinting used for?

Some of the applications of DNA fingerprinting techniques include: murder cases, rape cases,

paternity testing, diagnosis of inherited disorders, military identification, and molecular

archaeology.

In this chapter, we will explore each application, as well as simplify DNA for the average

person, show how to run a fingerprint, trace its growth and impact on society, and look

toward future uses such as national DNA databases and DNA fingerprints as one day

becoming our National Identification Cards.

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Fig:-The clues to murder mysteries and crime are found in the minute evidence of blood

and body fluids. The solution to many crimes is found in DNA 'fingerprinting'

1.6 WHAT ARE DNA FINGERPRINTS?

DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is a

technique employed by forensic scientists to assist in the identification of individuals by their

respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's

DNA makeup, which can also be used as the person's identifier.

Although 99.9% of human DNA sequences are the same in every person, enough of the DNA

is different to distinguish one individual from another. DNA profiling uses repetitive

("repeat") sequences that are highly variable, called variable number tandem repeats (VNTR).

VNTRs loci are very similar between closely related humans, but so variable that unrelated

individuals are extremely unlikely to have the same VNTRs.

1.7 Principles of DNA fingerprinting:

1.7.1 Base pairing;-

Each type of base on one strand forms a bond with just one type of base on the other strand.

This is called complementary base pairing. Here, purines form hydrogen bonds to

pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two

nucleotides binding together across the double helix is called a base pair. As hydrogen bonds

are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA

in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or

high temperature. As a result of this complementarity, all the information in the double-

stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA

replication. Indeed, this reversible and specific interaction between complementary base pairs

is critical for all the functions of DNA in living organisms.

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Top, a GC base pair with three hydrogen bonds. Bottom, an AT base pair with two hydrogen

bonds. Non-covalent hydrogen bonds between the pairs are shown as dashed lines.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two

hydrogen bonds, and GC forming three hydrogen bonds (see figures, left). DNA with high

GC-content is more stable than DNA with low GC-content, but contrary to popular belief,

this is not due to the extra hydrogen bond of a GC base pair but rather the contribution of

stacking interactions (hydrogen bonding merely provides specificity of the pairing, not

stability).

CHAPTER2:“METHODS OF DNA FINGERPRINTING”

RFLP:

Restriction fragment length polymorphism (RFLP) analyzes the length of the strands of the

DNA molecules with repeating base pair patterns. DNA molecules are long strands found

tightly wound in chromosomes which are contained in the nucleus of each human cell.

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Within each DNA strand are numbers of genes that determine the particular characteristics of

an individual. While about 5% of the gene compositions on DNA contain this type of genetic

information, the other 95% do not. However, of the 95%, these non-coding genes contain

identifiable repetitive sequences of base pairs, which are called Variable Number Tandem

Repeats (VNTR). To extract a DNA fingerprint, a Southern blot is performed and the DNA

is analyzed via a radioactive probe. The restriction fragment length polymorphism analysis is

used to detect the repeated sequences by determining a specific pattern to the VNTR, which

becomes the person's DNA fingerprint. The drawback with this system is that it requires a

considerable amount of DNA in order to be used.

PCR

The polymerase chain reaction (PCR) was developed by Karry Mullis of the Cetus

Corporation in 1983 for use in research laboratories for establishing hereditary

authentication. The PCR analysis amplifies the DNA molecules using a smaller sample. On

the forensic front, the PCR found to be useful in identifying DNA fingerprints in criminal

matters and in paternity tests because it requires less amounts of DNA because it makes

identical copies of the DNA sample. The PCR analysis amplified isolated regions on the

strands of the DNA under examination. The drawback was that it was not as discriminating

as the RFLP.

AmpFLPAmplified fragment length polymorphism (AmpFLP) came into vogue in the 90's and is still

popular in the smaller countries involved in the process of DNA fingerprinting. It remains

attractive because of its relatively less complicated operation and the cost-effectiveness of

the procedure. By using the PCR analysis to amplify the minisatellite loci of the human cell,

this method proved quicker in recovery than the RFLP. However, due to the use of gel in its

analysis phase, there are issues of bunching of the VTRN's, causing misidentifications in the

process.

STRThe short tandem repeat (STR) methodology for extracting DNA is the system most widely

used form of DNA fingerprinting. This system is based on the features of PCR, as it utilizes

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specific areas that have short sequential repeat DNA. The STR analyzes how many times

base pairs repeat themselves on a particular location on a strand of DNA. The big advantage

in this method is that the DNA comparisons can match the possibilities into an almost

endlessrange.

DNA fingerprinting has been extremely successful for use in the personal identification of

criminal suspects, paternity issues, as well as in identification of the deceased. DNA,

however, still poses issues because the VNTRs are not evenly distributed in all people

because they are inherited. In addition, there is still the human element as the final voice in

the administration of all DNA fingerprinting procedures. However, as forensic science

continues its work in DNA testing, there appears to be no limit to the value it can render to

society.

2.1Process of Making a DNA Profile using RFLP method:-

The key to DNA profiling is to make a comparison of unique loci of the DNA left at the

crime scene with a suspect’s DNA. The portion of the genome where there is a lot of

diversity among individuals is called polymorphic regions. The polymorphic regions used for

forensics are the non-coding regions. These are the regions of the DNA that do not code for

proteins and they make-up 95% of our genetic DNA. These regions are therefore called the

“junk” portion of the DNA. Although these “junk” regions do not generate proteins, they can

regulate gene expression, they aid in the reading of other genes that do formulate the proteins,

and they are a large portion of the chromosome structure (How DNA Evidence Works, 2004).

The non-coding DNA regions are made-up of length polymorphisms, which are variations in

the physical length of the DNA molecule. The DNA profile analyzes the length

polymorphisms in the non-coding areas. These polymorphisms are identical repeat sequences

of DNA base pairs. The number of tandem repeats at specific loci on the chromosome varies

between individuals. For any specific loci, there will be a certain number of repeats. These

repeat regions are classified into groups depending on the size of the repeat region. Variable

number of tandem repeats (VNTRs) have repeats of 9-80 base pairs. Short tandem repeats

(STRs) contain 2-5 base pair repeats (Brief Introduction to STRs, 2004). For each of our 23

pairs of chromosomes, we inherit one copy of each chromosome from our mother and the

other from our father. “This means that you have two copies of each VNTR locus, just like

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you have two copies of real genes (Figure-4). If you have the same number of sequence

repeats at a particular VNTR site, you are called homozygous at that site; if you have a

different number of repeats, you are said to be heterozygous” (How DNA Evidence Works,

2001).

Figure 4: The diagram above displays three possible VNTR combinations.

DNA analysis is a laboratory procedure that requires a number of steps. There are a number

of techniques used by different laboratories, however in this paper two techniques will be

reviewed: Restriction Fragment Length Polymorphism (RFLP), and Polymerase Chain

Reaction (PCR) using Short Tandem Repeats (STRs).

The first step in both procedures involves the extraction and purification of the DNA. Before

a DNA sample can be analyzed, the DNA needs to be isolated from the other organic and

non-organic portions of the sample. The type of sample will determine the technique used to

isolate the DNA. The sample may be boiled with a detergent that breaks down the proteins

and other cellular material but does not affect the DNA. Enzymes may be added to break

down proteins and other cellular material. Organic solvents may be used to separate the DNA

from the other organic and non-organic material. The DNA is then separated from the

proteins and othercellular material (DNA Forensics, Problem Set 1, 2004).

2.1.1Steps for DNA fingerprinting by RFLP method

1. Isolation of DNA.

2. Cutting, sizing, and sorting.

3. Transfer of DNA to nylon.

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4. Probing.

5. DNA fingerprint.

Fig : showing the basic steps of the dna fingerprinting

Step 1: Isolation of DNA:-

The process begins with a sample of an individual's DNA (typically called a "reference

sample"). The most desirable method of collecting a reference sample is the use of a buccal

swab, as this reduces the possibility of contamination. When this is not available (e.g.

because a court order may be needed and not obtainable) other methods may need to be used

to collect a sample of blood, saliva, semen, or other appropriate fluid or tissue from personal

items (e.g. toothbrush, razor, etc.) or from stored samples (e.g. banked sperm or biopsy

tissue). Samples obtained from blood relatives (biological relative) can provide an indication

of an individual's profile, as could human remains which had been previously profiled.

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DNA can be extracted from almost any human tissue.

Buccal cells from inside cheek for paternity tests.

Sources of DNA at crime scene: blood, semen, hair follicle, saliva.

DNA extracted from evidence is compared to DNA from known individuals.

Step2:Cutting, sizing, and sorting

Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in

DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction

enzymes play a very important role in the construction of recombinant DNA molecules, as is

done in gene cloning experiments. Another application of restriction enzymes is to map the

locations of restriction sites in DNA. Special enzymes called restriction enzymes are used to

cut the DNA at specific places. For example, an enzyme called EcoR1, found in bacteria, will

cut DNA only when the sequence GAATTC occurs. The DNA pieces are sorted according to

size by a sieving technique called electrophoresis. The DNA pieces are passed through a gel

made from seaweed agarose (a jelly-like product made from seaweed). This technique is the

DNA equivalent of screening sand through progressively finer mesh screens to determine

particle sizes.

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Fig:- showing the digestion of dna fragment by the restriction enzymes

Sepration of dna fragments by GEL ELECTROPHORESIS

according to their size:

Agarose gel electrophoresis is a method used in biochemistry and molecular biology to

separate a mixed population of DNA and RNA fragments by length, to estimate the size of

DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are

separated by applying an electric field to move the negatively charged molecules through an

agarose matrix. Shorter molecules move faster and migrate farther than longer ones because

shorter molecules migrate more easily through the pores of the gel. This phenomenon is

called sieving. Proteins are separated by charge in agarose because the pores of the gel are

too large to sieve proteins.

"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules

through the gel matrix. By placing the molecules in wells in the gel and applying an electric

field, the molecules will move through the matrix at different rates, determined largely by

their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if

negatively charged or toward the cathode if positively charged.

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Materials required for agarose gel electrophorosis

Typically 10-30 μl/sample of the DNA fragments to separate are obtained, as well as a

mixture of DNA fragments (usually 10-20) of known size (after processing with DNA size

markers either from a commercial source or prepared manually).

Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0 .

Agarose .

An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O). The stock

solution be careful handling this. Alternative dyes may be used, such as SYBR Green.

Nitrile rubber gloves. Latex gloves do not protect well from ethidium bromide .

A color marker dye containing a low molecular weight dye such as "bromophenol

blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make

the DNA solution denser so it will sink into the wells of the gel).

A gel rack.

A "comb" .

Power Supply .

UV lamp or UV lightbox or other method to visualize DNA in the gel .

Preparation

There are several methods for preparing gels. A common example is shown here. Other

methods might differ in the buffering system used, the sample size to be loaded, the total

volume of the gel (typically thickness is kept to a constant amount while length and breadth

are varied as needed). Most agarose gels used in modern biochemistry and molecular biology

are prepared and run horizontally.

1. Make a 1% agarose solution in 100ml TAE, for typical DNA fragments (see figures).

A solution of up to 2-4% can be used if you analyze small DNA molecules, and for

large molecules, a solution as low as 0.7% can be used.

2. Carefully bring the solution just to the boil to dissolve the agarose, preferably in a

microwave oven.

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3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or

swirl the solution while cooling.

Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety

see ethidium bromide

1. Add 5 µl ethidium bromide (from a stock solution of 10 mg/ml) per 100 ml gel

solution for a final concentration of 0.5 µg/ml. Be very careful when handling the

concentrated stock. Some researchers prefer not to add ethidium bromide to the gel

itself, instead soaking the gel in an ethidium bromide solution after running, in this

case the ethidium bromide solution has final concentration of 0.5 µg/ml.

2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.

3. Insert the comb at one side of the gel, about 5–10 mm from the end of the gel.

4. When the gel has cooled down and become solid, carefully remove the comb. The

holes that remain in the gel are the wells or slots.

5. Put the gel, together with the rack, into a tank with TAE. Ethidium bromide at the

same concentration can be added to the buffer. The gel must be completely covered

with TAE, with the slots at the end electrode that will have the negative current.

Procedure

After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a

DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and

apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA

ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When

the "front wave" approaches the end of the gel, the current is stopped. The DNA is stained

with ethidium bromide, and is then visible under ultraviolet light.

1. The agarose gel with three slots/wells (S).

2. Injection of DNA ladder (molecular weight markers) into the first slot.

3. DNA ladder injected. Injection of samples into the second and third slot.

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4. A current is applied. The DNA moves toward the positive anode due to the negative

charges on its phosphate backbone.

5. Small DNA strands move fast, large DNA strands move slowly through the gel. The

DNA is not normally visible during this process, so the marker dye is added to the

DNA to avoid the DNA being run entirely off the gel. The marker dye has a low

molecular weight, and migrates faster than the DNA, so as long as the marker has not

run past the end of the gel, the DNA will still be in the gel.

6. Add the color marker dye to the DNA ladder

After the electrophoresis is complete, the molecules in the gel can be stained to make

them visible. Ethidium bromide, silver, or Coomassie Brilliant Blue dye may be used for

this process. Other methods may also be used to visualize the separation of the mixture's

components on the gel. If the analyte molecules fluoresce under ultraviolet light, a

photograph can be taken of the gel under ultraviolet lighting conditions, often using a Gel

Doc. If the molecules to be separated contain radioactivity added for visibility, an

autoradiogram can be recorded of the gel.

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Fig:- Gel electrophoresis result

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Step 3; Transfer of dna fragment from gel to nitrocellulose

membrane:-

A Southern blot is a method routinely used in molecular biology for detection of a

specific DNA sequence in DNA samples.

The original method was first developed by E. M. Southern in 1975 and later was modified

by various people for various applications. The process of Southern blotting starts after the

agarose gel electrophoresis of restriction digest or DNA. The gel is first washed with buffer

to remove the broken or fragmented residues of agarose formed during banding and the

accumulated contaminants.

Then the gel is placed in acid solution (0.25 M HCI) for about 5-10 min. During this step, the

DNA undergoes depurination. After this step the gel is placed in 0.25 M NaOH alkali

solution. This step ensures that the DNA is shorter in length (<500 bp) and is in single-

stranded form. DNA fragments large in size (>10 kb) require a longer time and move in zig-

zag form when compared to shorter DNA fragments.

Finally, the gel is extensively washed with buffer to remove the traces of HCI and NaOH.

Then the gel is placed on the filter paper with wigs dipped in a reservoir containing transfer

buffer placed in transfer buffer reservoir tank which is placed on the glass plate.

Nitrocellulose or nylon filter paper is placed on the gel above which a stack of blotting

papers soaked in transfer buffer is placed and gently pressed using glass plate or rod to

remove the air that is trapped between the gel and membrane. Then a stack of unsoaked

blotting papers is placed above them and the glass plate with a weight of 500 g is placed

above it.

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By capillary action the transfer buffer is transferred from the reservoir to the blotting papers.

During this process the DNA from the gel is transferred onto the nitrocellulose paper and

immobilized there due to the negative charge of the DNA and the positive charge of the

membrane.

This set up is allowed to stand like that for 12-18 hours. After this time period the

nitrocellulose or nylon membrane is taken out from the set up carefully. As the binding of the

DNA to the nitrocellulose or nylon membrane is weak, it has to be fixed more strongly, so

that the DNA is not washed off during the washing of the membranes. There are two widely

used methods for this procedure.

After the transfer, the membrane is exposed to ultraviolet rays, so that a bond is formed

between thymine residues present in the DNA and the positively charged amino groups on

the surface of the nylon membrane. This method is called as UVcross linking method. The

other method is baking of the nitrocellulose or nylon membrane at 80°C in a vacuum oven.

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Step 4: Probing

Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA

fingerprint. Each probe typically sticks in only one or two specific places on the nylon sheet.

In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable

length (usually 100-1000 bases long), which is used in DNA or RNA samples to detect the

presence of nucleotide sequences (the DNA target) that are complementary to the sequence in

the probe. The probe there by hybridizes to single-stranded nucleic acid (DNA or RNA)

whose base sequence allows probe-target base pairing due to complementarity between the

probe and target. The labeled probe is first denatured (by heating or under alkaline conditions

such as exposure to sodium hydroxide) into single DNA strands and then hybridized to the

target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in

situ.

Let's start with the mechanism, how the probe identifies the sequence. Normally DNA is in

double stranded form, and the nucleotides A-T and G-C will form 2 and 3 hydrogen bonds to

each other (A to T and C to G). When we heat the DNA, it will denaturate to single stranded

form and hydrogen bonds will no more bond the strands together, so we will have two

complementary strands.

Now, lets add a short piece of single stranded DNA which is labeled with radioactive

phosphorus or sulfur*. If the probe's sequence is ATTCCGG...TT, it will slide past DNA

which doesn't have complementary sequence. When our probe happens to hit the sequence

AA...CCGGAAT, it will attach firmly to its "missing half". Of course most probe molecules

will never find their complementary sequences and will attach to TT...CCCGAAT, for

example. But we can adjust the environment so that those probes that are not firmly attached

will break off, and only the true sequences are found. This separation can be done by

adjusting temperature and salt consentration etc., collectively referred to as the "stringency".

In Southern blotting we have first separated genomic DNA from a tissue sample. Then the

long genomic DNA is cut into smaller parts with restriction enzymes. Pieces are separated

according to their size with agarose gel electrophoresis and then transferred to a membrane,

usually nylon or nitrocellulose.

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Now that we have our DNA bound to a solid surface, separated by size, we denaturate the

DNA and add the radiolabeled probe. The probe attaches to it's counterpart, some to almost

their counterpart, and some just wander over the membrane. Then we just wash away the

probes which are loosely attached to DNA, because the more complementary the sequence is,

the more hydrogen bonds can form and the stronger the "union" that will form between these

two molecules. And after washing we just expose the membrane to x-ray film and can see, if

we, for example, were succesfull to achieve a transgenic calf.

2.1.2ANALYSIS OF DNA FINGERPRINTS:

DNA fingerprinting is often used in criminal cases to determine a suspect's guilt. DNA from

a blood, hair root, or semen sample found at the scene of a crime can prove guilt or innocence

with high precision. The DNA sample is cleaved with a restriction enzyme and the resulting

fragments are separated using Southern blotting techniques. DNA from the crime scene is

analyzed on the same gel as DNA from the potential criminals, and therefore, DNA collected

from the scene can be compared with that from various suspects and the RFLP's produced

from the DNA of the guilty suspect will match with the RFLP's produced from DNA

collected from the crime scene.

In the following example, a woman was raped while she and her fiance were sleeping in their

car. They were found the next morning in the woods next to a recreation area and both had

died of gunwounds. One man was later found driving the stolen vehicle and he told

authorities of the friend that was with him the night of the murders. In order to determine

which suspect was guilty of raping the woman, DNA fingerprinting (RFLP analysis) was

used. DNA from a semen sample retrieved from the body was compared with DNA from

blood samples from both suspects. These DNA samples were cut with a particular restriction

enzyme and fragments were separated by gel electrophoresis. Figure 3 shows the RFLP's

from the semen sample in yellow and the RFLP's from both suspects in purple and blue. It is

shown in this figure that suspect 2 is the man that raped this woman. It is possible that an

innocent person could show the exact same restriction fragments, but the chance of this is 1

in 9,390,000,000, which is twice the human population of the world! Suspect 2 was conviced

of rape and murder and received a double death sentence. This happened to be the first case

in the world in which the conviction of the death sentence was based on DNA fingerprinting

(The Dolan DNA Learning Center, 2000).25

 

Fig:- RFLP analysis of the semen sample collected from the raped woman versus blood

samples of two suspects. The pink and blue fragments were produced from genomic DNA

from the suspects and the yellow fragments were produced from genomic DNA from the

victim. This figure was reproduced pending permission by the

2.2“ AFLP-PCR:Amplified Fragment Length Polymorphism ”

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Amplified Fragment Length Polymorphism PCR (or AFLP-PCR or just AFLP) is a

PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic

engineering. Developed in the early 1990s by Keygene, AFLP uses restriction enzymes to

digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction

fragments. A subset of the restriction fragments is then selected to be amplified. This

selection is achieved by using primers complementary to the adaptor sequence, the restriction

site sequence and a few nucleotides inside the restriction site fragments (as described in detail

below). The amplified fragments are visualized on denaturing polyacrylamide gels either

through autoradiography or fluorescence methodologies.

AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique

was originally described by Vos and Zabeau in 1993. In detail, the procedure of this

technique is divided into three steps:

1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of

restriction half-site specific adaptors to all restriction fragments.

2. Selective amplification of some of these fragments with two PCR primers that have

corresponding adaptor and restriction site specific sequences.

3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of

the band pattern.

A variation on AFLP is cDNA-AFLP, which is used to quantify differences in gene

expression levels.

Another variation on AFLP is TE Display, used to detect transposable element mobility.

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FIG:-Example of AFLP Data from a Capillary Electrophoresis Instrument

2.3 Polymerase Chain Reaction (PCR)

PCR is a new technique that was developed in 1986 by Kary Mullis and was applied to

forensics in 1991 (Forensic Fact Files DNA Profiling, 2004). PCR has the ability to replicate

genetic material, and even proofread and make corrections on the copies (Forensic Fact Files

DNA Profiling, 2004). It involves the repeated copying of specified areas of DNA molecules.

These areas are the alleles and are specific sequences of base pairs. These target areas are the

variable regions. At either end of these target areas are “flanker” bases that are the non

variable regions. The flankers occur at the same locations on the chromosomes of all people.

The basis of PCR is to reproduce thousands of copies of a particular variable region of the

DNA that is of interest. This results in DNA fragments that vary in length due to the

variability in the number of repeated regions. The process is used to amplify a small amount

of DNA taken from a sample of blood, hair, etc. With each cycle of copying, the quantity of

the target allele is doubled resulting in an exponential amplification of the gene in the PCR.

After 30 copying cycles, there will be one billion times more copies of the target alleles than

at the start of the amplification process (Principle of the PCR, 1999).

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Figure : Steps in PCR. (Principle of PCR, 2004

29

There are three steps in the PCR process (see Figure):

1) Denaturation: This process takes place at 94 degrees Celcius. This stage separates the

DNA double helix, and forms DNA single strands. This also forces every enzymatic reaction

to come to a halt.

2) Annealing: This usually takes place at 54 degrees Celsius. The primers bind to their

complementary bases on the now single-stranded DNA.

3) Extension: This takes place at 72 degrees Celsius. This step involves the synthesis of

DNA by polymerase. Starting from the primer, the polymerase reads the template strand and

matches it with complementary bases. The result is two new helixes, each composed of one

of the original strands plus its newly assembled complementary strand (Principle of the PCR,

1999).

In order to obtain more copies of DNA, the three step process is repeated. Each phase only

takes 1-3 minutes, thus if you completed the process for 45 minutes, millions of copies could

be generated because every time the process is repeated the amount of DNA doubles

(exponentially) (Figure-8).

FIG: Diagram of Exponential Growth in the Number of DNA Copies During PCR.

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2.3.1DNAFingerprintingAnalysisThis DAF analysis system is used routinely to characterise unknown isolates of Foc from

Australia and worldwide by comparing the DNA fingerprints of these unknown isolates with

those from our DNA reference collection (which spans all the currently identified VCGs of

Foc). This DAF analysis system has been thoroughly optimised and provides an informative

and reproducible platform for the molecular characterisation of unknown isolates of Foc.

Genome-specific DNA fingerprint patterns are analysed and scored manually (by eye);

fingerprints of test samples are compared to standards (characterised isolates) by

assessing the general fingerprint pattern and scoring for presence/absence of polymorphic

bands (see Figure 8). Scoring is made easier by placing gels on a lightbox, which enables the

discrimination of lightly stained bands. Test samples can be compared with standards from

other gels by overlapping gels on the lightbox, or by digitally cutting and pasting the lanes

next to one to build a new image that makes scoring easier. DAF analysis is used in

conjunction with VCG testing for the characterisation of unknown isolates of Foc.

Figure 8. DNA Fingerprinting analysis of a range of Australian and overseas VCGs of Foc,

visualised by polyacrylamide gel electrophoresis and silver staining. The DAF PCR was

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performed using the HIRH single oligonucleotide primer. This DAF analysis clearly shows

the utility of this technique for differentiating closely related VCGs of Foc. Isolate

information is annotated on the gel photograph. Image courtesy of the CRC for Tropical

Plant Protection.

Analysis of the alleles uses length variation of the amplified alleles. The alleles used are

repeated units of the same sequence of bases. The length variation is the number of times that

a particular base sequence is repeated between flankers. PCR techniques are divided into

different categories depending on the number of base pairs in the repeated units. Variable

Number of Tandem Repeats (VNTR) uses between 9 and 40 bases per unit; Amplified

Fragment Length Polymorphism (AmpFLP) uses between 8 and 16 bases per unit; and Short

Tandem Repeats (STR) uses between 4 and 6 bases per unit (Schumm, 1996). STRs will be

focused on in this paper because it is the technique of choice for most forensics laboratories

presently.

2.4.“STRs(short tenden repeats)”

The short tandem repeat (STR) methodology for extracting DNA is the system most widely

used form of DNA fingerprinting. This system is based on the features of PCR, as it utilizes

specific areas that have short sequential repeat DNA. The STR analyzes how many times

base pairs repeat themselves on a particular location on a strand of DNA. The big advantage

in this method is that the DNA comparisons can match the possibilities into an almost

endlessrange.

During forensic examination, an STR with a known repeat sequence is extracted and

separated by electrophoresis. The distance that the STR migrated is examined. However,

capillary columns are now used rather than the gel electrophoresis that was used in the RFLP

analysis. Two short pieces of synthetic DNA called primers are specially designed to attach

to a conserved common non-variable region of DNA, which flanks the variable target region

of the DNA. A mixture of chemicals including the primers, the individual bases and a

copying enzyme (polymerase) is added to the solution with the original DNA strand that is to

be copied. The primers are short chains of the bases that make up the single strand of DNA.

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Primers are complementary to the conserved regions flanking the targeted STR (black arrows

in Figure-6). The primer used in the PCR amplification process attaches a fluorescent tag to

the target alleles enabling the distance traveled by the DNA fragments during capillary

electrophoresis to be detected using a fluorescent scanner (Blackett Family DNA Activity 2,

2004).

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CHAPTER 3: “APPLICATIONS OF DNA

FINGERPRINTING”

3.1 DNA FORENSICS:

Forensic DNA analysis has been admitted into United States courtrooms since 1987. The

common question being answered is “Who is the source of this biological material?” (Inman

and Rudin, 1997). DNA profiling was first used in the United States courtroom in the case of

Tommie Lee Andrews. (He was suspected of serial rape, therefore, the investigators wanted

to link his DNA to the DNA of the 23 victims. Because of the DNA analysis technology and

the CODIS system, Andrews was linked to the series of rapes, and sentenced to 115 years

(Ramsland, 2004). Profiling has helped to acquit or convict suspects of rape and/or murder

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however it is used in less than one percent of all criminal cases (Genetic Science Learning

Center, 2004). DNA profiling does not claim to be an absolute identification, but may be very

strong evidence and is considered to be just one part of the entire case. DNA profiling is used

primarily in sexual assault cases. Prior to DNA testing, labs were limited regarding the

amount of genetic information that could be obtained from semen. Now with DNA testing,

the genetic content of the sperm itself can be examined. The RFLP method of analysis needs

a considerable amount of material for testing and may be unable to determine anything from

a sample that has any degradation. The PCR analysis needs only a minute amount of sample

because it replicates the DNA and can work even with degraded samples (Sullivan Personal

Interview 2004). In the case of a rape investigation four profiles are formed (Figure-10): the

first is a DNA profile from the blood of the victim, the second DNA profile is formed from

the blood of the defendant, thirdly a vaginal swab is taken, and the female and male fractions

are separated and profiled separately. The victim’s blood profile and the vaginal swab profile

should match alleles. The defendant’s blood sample profile should match the male fraction

profile in order to consider him as a suspect. It is important to remember that DNA profiling

does not incriminate a suspect it can only exclude suspects.

Some examples of DNA uses for forensic identification include:

• Identify potential suspects whose DNA may match evidence left at crime scenes

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• Exonerate persons wrongly accused of crimes

• Identify crime and catastrophe victims

• Establish paternity and other family relationships

• Identify endangered and protected species as an aid to wildlife officials (could be used for

prosecuting poachers)

• Detect bacteria and other organisms that may pollute air, water, soil and food

• Match organ donors with recipients in transplant programs

• Determine pedigree for see or livestock breeds

• Authenticate consumables such as caviar and wine

(The FBI’s DNA Databasing Initiatives, 2000)

Murder or Rape Applications

DNA Fingerprinting has evolved to take part in a plethora of different applications. Thefirst,

and most well known application is forensics, either murder or rape cases. Suppose anofficer

arrived at a crime scene of a murder, and the only evidence that could be found was a

bloodstain on the carpet from the suspect. If you were put on a jury and told, based on

scientific evidence using DNA Fingerprinting, that this suspect was the murderer, would you

convict him? In the beginning stages of its existence, courts would not always allow the

evidence to be admitted, based on the fact it hadn't been completely proven to be accurate. As

you'll see in a following chapter, many court cases and laws continued to set precedence, and

DNA Fingerprinting eventually became more and more accessible and less controversial in

the court of law. The FBI began to establish DNA Fingerprinting as means of connection to

crimes early on. Between the years of 1989 and 1996, they used the technique in about

10,000 sexual assault cases (Establishing Innocence, 2004). In those cases, 2,000 prime

suspects were proven innocent based on the results of the DNA profiling (Establishing

Innocence, 2004). In Figure 8, you can see an example of an RFLP fingerprint analysis of 7

suspects relative to a crime scene bloodstain. It seems very clear that Suspect 3 was present at

the crime scene. It is also true to say that without this method of testing, those 2,000 innocent

prime suspects could most likely have faced conviction (Establishing Innocence, 2004).

Many cases from decades ago have been recently reopened and their verdicts changed by

36

admitting new DNA evidence. Many of these cases will be explained in depth in a later

chapter.

Figure 20 – Evidence that is now used thanks to DNA Fingerprinting techniques.(Huskey, 1995)

3.2 Paternity Testing

Paternity testing has also become a very commonly used application of DNA Fingerprinting.

When a child is born, the child inherits 23 chromosomes from the mother, and chromosomes

from the father. Therefore, when a DNA test is done, “the visible band pattern of the child is

unique. Half matches the mother and half matches the father”. A DNA paternity test is the

most accurate form of testing possible to determine parentage. If the patterns do not match on

two or more probes, then the father is 100% excluded, which means he is without question

not the father. If a match is present on every DNA probe, the probability of paternity is 99.9%

or greater. In a Court of Law, 99% is accepted as proof of paternity (DNA Diagnostic Center,

2004). In Figure 9, you can see that the mother (blue bands) and father (orange bands) both

37

have separate DNA patterns. Now look at daughter 1. You can see that some of the mothers

same blue bands are inherited as well as some of the fathers orange bands. Daughter 2 has the

mothers blue bands, but not the orange band, instead 38 red bands. This proves that the father

of daughter 1 is not the father of daughter 2, but a father from the mother’s previous

marriage. Son 1 is a child of both of the shown parents as well. Son 2, however, is adopted

because he doesn't have any of the parents DNA. One of the most famous paternity cases

involved Thomas Jefferson, the third President of the United States, who in 1802, was

accused of impregnating Sally Hemings, a slave at the Jefferson estate. No verification or

conviction was ever brought about however. The story was sustained throughout the decades,

and in 1998, Dr. Eugene Foster and a team of geneticists, conducted DNA tests to prove the

accuracy of these accusations. The test results proved that it was indeed a Jefferson who

fathered Sally Heming's son, Eston. At the time of the child’s birth, there where

approximately twenty-five Jefferson's living in Virginia, all of who carried the

chromosome that would match the child's. The verdict proved that it was probable, yet not

conclusive, that Thomas Jefferson was the father of Eston Hemings (The Plantation, 2004).

38

Figure 21 – A DNA Paternity Test showing results after being tested using DNA Fingerprints. (Antler,

2004)

3.3 MIA Soldiers:

Another application of DNA Fingerprinting is for historical clarifications and identifications.

Over the last decade, hundreds of missing in action (MIA) soldiers, that were never

identified, have been identified through the use of DNA Fingerprinting. How is this possible?

Relatives of unidentified soldiers who are willing to use a sample kit to submit a blood and

saliva sample to the laboratory, where “forensic anthropologists, forensic dentists and

equipment recovery specialists who work at the U.S. Army Central Identification

Laboratory” (Tippy, 2004) analyze the sample, and are able to match DNA with MIAs, with

as little as a bone fragment recovered from a killed soldier (Tippy, 2004).

"The most famous American memorial for unidentified soldiers killed in combat is located at

Arlington National Cemetery in Arlington, Virginia" (Unknown Soldier). It has inherited the

name as the Tomb of the Unknown Soldier (Unknown Soldier, 2004). In 1998, the remains of

soldiers killed during the Vietnam War, which occurred between 1959 and 1975, were

removed for hope of identification. After testing was completed, the remains were identified

as First Lieutenant Michael Blassie, an Air Force pilot who was killed when his helicopter

was shot down in Vietnam in 1972. After the positive identification, his family buried the

remains in Saint Louis, near Blassie's childhood home. In 1999, Pentagon officials made the

decision that based on technological advances in this DNA profiling and the ability to

identify MIA's, no other soldier from the Vietnam War would be buried in the Tomb of the

Unknown Soldier (Unknown Soldier, 2004).

3.4 Inherited Disorder Testing

DNA Fingerprinting is also capable of determining inherited disorders in adults, children, and

unborn babies. A historical example to prove this involves our sixteenth president and the

issue of Marfan's Syndrome (Betsch, 1994), a disease which is characterized by unusually

39

long and nimble limbs. As you know, President Lincoln was abnormally tall during his

lifetime which lasted from 1809 to 1865. Could DNA evidence from Lincoln be recovered

that would prove this disease existed? According to David F. Betsch, "the technology is so

powerful that even the blood-stained clothing from Abraham Lincoln has been analyzed for

evidence of a genetic disorder called Marfan's Syndrome" (Betsch, 1994). Although many

attempts have been made to clearly identify this disorder in Abraham Lincoln, no conclusive

result has been reached due to damaging of historical artifacts. This genetic testing allows

people to "test for some 40 anomaly that flags a disease or disorder” (Inherited Disorders,

2002). “Much of the current excitement in gene testing, however, centers on predictive gene

testing: tests that identify people who are at risk of getting a disease, before any symptoms

appear” (Inherited Disorders, 2002).

3.5 Personal Identification

DNA fingerprinting has seen tremendous growth over the last couple of decades, but is there

more on the horizon for new applications? One of the main challenges in the field is to create

an ideal system of personal identification that would be “indelible, unalterable and –unlike an

ID card –part of the individual” (Marx, 1998). The scientific goal is to generate this as a

national standard, where every citizen is included within the database, and to prove that there

are no drawbacks, such as misuse of medical information during the identification process, or

any cautious steps that are taken before DNA fingerprinting becomes the standard (Marx,

1998). In 2002, Alec Jeffreys, the inventor of DNA fingerprinting, put forth his theory on the

DNA databases. He believed that “every citizen’s genetic information should be stored on the

UK national register” (O'Brien, 2002). Jeffreys went on to say that “if we’re all on the

database, we’re all in exactly the same boat – the issue of discrimination disappears”

(O'Brien, 2002). The advantages and disadvantages of DNA databases, as well as its progress

in the modern day society, will be explained in more depth in a following chapter. Another

challenge in this growing field is to simplify and hasten the procedure of running a DNA

fingerprint which will revolutionize forensics. In Australia, researchers have claimed that

they have a technique that is twice as fast as conventional DNA technology and is capable of

cranking out 1,000 samples a day, where most laboratories manage only a few dozen

40

(Dayton, 2002). “Moreover, profiles are derived from DNA contained in a single cell —

existing 41 procedures require at least 500 cells” (Dayton, 2002). Team leader, Ian Findlay,

of the Australian Genome Research Facility, said of his team’s recent discoveries, that “it’s

such a breakthrough we’re waiting for the rest of the world to catch up.” (Dayton, 2002) So

this is where the debate begins. In the chapters to follow, forensics of DNA, landmark DNA

court cases, and DNA databases will be examined. Also issues such as the validity of these

tests, and the use within the courtroom and why some courts still will not completely trust

DNA evidence in the courtroom.

3.6 DNA Forensics Databases

National,DNADatabank:CODIS:

The COmbined DNA Index System, CODIS, blends computer and DNA technologies into a

tool for fighting violent crime. The current version of CODIS uses two indexes to generate

investigative leads in crimes where biological evidence is recovered from the crime scene.

The Convicted Offender index contains DNA profiles of individuals convicted of felony sex

offenses (and other violent crimes). The Forensic index contains DNA profiles developed

from crime scene evidence. All DNA profiles stored in CODIS are generated using STR

(short tandem repeat) analysis.

CODIS utilizes computer software to automatically search its two indexes for matching DNA

profiles. Law enforcement agencies at federal, state, and local levels take DNA from

biological evidence (e.g., blood and saliva) gathered in crimes that have no suspect and

compare it to the DNA in the profiles stored in the CODIS systems. If a match is made

between a sample and a stored profile, CODIS can identify the perpetrator.

This technology is authorized by the DNA Identification Act of 1994. All 50 states have laws

requiring that DNA profiles of certain offenders be sent to CODIS. As of January 2003, the

database contained more than a million DNA profiles in its Convicted Offender Index and

about 48,000 DNA profiles collected from crime scenes but which have not been connected

to a particular offender.

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As more offender DNA samples are collected and law enforcement becomes better trained

and equipped to collect DNA samples at crime scenes, the backlog of samples awaitning

testing throughout the criminal justice system has increased dramatically. In March 2003

President Bush proposed $1 billion in funding over 5 years to reduce the DNA testing

backlog, build crime lab capacity, stimulate research and development, support training,

protect the innocent, and identify missing persons. For more information, seethe U.S.

Department of Justice's.

Potential Benefits of DNA Databanking Arrestee:

Most major crimes involve people who also have committed minor offenses.

Innocent people currently are incarcerated for crimes they did not commit; if samples

had been taken at the time of arrest, these individuals would have been excluded early

in the investigative process.

Moving the point of testing from conviction to arrest would result in savings in

investigation, prosecution, and incarceration.

Investigators would be able to compare other cases against the arrested person's DNA

profile, just as with fingerprints.

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CHAPTER 4: CONCLUSION

This report has been an effort to bring to the public a greater understanding of DNA

fingerprinting and its uses. To begin our examination we looked at DNA structure and the

process of creating a DNA profile. To continue with the paper we explored the stringent steps

that must be taken to collect and preserve forensic evidence to help its acceptance in the

courtroom. It logically followed that we should inspect the historical significance of the

precedence set by landmark court cases concerning the admissibility of technical evidence

including DNA profiles. Finally we looked towards the future with the establishment and

ongoing legislative modifications of the CODIS system. We chose these subjects to focus on

because it is important to inform the general public of them in order to dispel any myths

regarding the usage of DNA in forensics. We felt that this topic provided a perfect example

of a new technology with great impact on society, worthy of an IQP. The advances in

forensic technology have allowed the sophistication and standardization of the process by

which forensic evidence is collected and preserved in order to increase the chances of its

acceptance in the courtroom. We have found that due to the nature of most forensic evidence,

heat and moisture can cause severe degradation to samples. Because of this it is necessary to

avoid packaging DNA evidence in plastic containers because plastic does not allow moisture

to escape, and the DNA degrades. Also, because contamination, especially in the case of PCR

analysis can render the resulting profile virtually useless, it is essential that only clean

instruments are used in the collection, and that care is taken to collect elimination control

samples. Chain of custody must also be established to avoid any argument of tampering. The

43

importance of the treatment of this genetic material cannot be overstressed as the world

painfully 77 learned in the OJ Simpson trial. Without the rigid guidelines that are now in

place, it would be difficult to argue the credibility of the evidence itself.

44