A Fingerprint of Our Genetic Makeup: DNA Typing

Deoxyribonucleic acid (DNA): A polymeric molecule consisting of deoxyribonucleotide building blocks that in a double-stranded, double helical form is the genetic material of most organisms. (courtesy of iGenetics)

DNA is what sets all organisms apart. The DNA that we have is what makes us humans and the DNA horses have is what makes them horses. Within humans as a species much of the DNA molecule is identical but no two people have the exact same genome and this is where DNA typing or fingerprinting comes into play. DNA typing techniques are used in criminal trials, paternity tests, population genetics studies, proving pedigree status for horses and dogs, and identification of dead bodies. Of course there are numerous other uses and some will be explored later on.

The coding regions of DNA (exons) are very similar between human individuals but there are portions of DNA which seem to be noncoding regions which we know as introns. It is in these regions that repeated sequences of base pairs exist which differ from one person to the next. These sequences are called Variable Number of Tandem Repeats (VNTRs) if they are five to a few tens of base pairs long. Short Tandem Repeats (STRs) are two, three or four base pairs long and also show great variation. Every individual has some VNTRs (and STRs) and their VNTRs come from the genetic information donated by their parents. The VNTRs can be inherited from either that person’s mother or father and can be a combination of both. A very important part of this is that a person’s VNTRs will never have sequences that their parents do not have. Scientists can use these regions (VNTRs) to make a “fingerprint” to determine whether two DNA samples are from the same person, related people, or non-related people.

In the image to the left it is shown that Daughter one and Son one have VNTRs from both their mother and father. Daughter two has her mother’s VNTRs but not her father’s meaning she is from the mom’s other marriage. Son two has neither mom nor dad’s VNTRs because that son was adopted.

The two main processes for DNA analysis are:

1. Using restriction fragment length polymorphisms (RFLPs) detected by restriction enzyme digestion and Southern blotting analysis.

2. Using Length polymorphisms detected by PCR amplification and a dot blot assay.

RFLP Identification

1. Isolate DNA from blood, semen, or any other cell sample.

2. Extract the DNA from the cell.

3. Digest the DNA into different sized fragments by restriction enzymes such as EcoR1.

4. Separate the DNA fragments by gel electrophoresis.

5. Denature the DNA strand into two single strands.

6. Transferring the DNA band pattern to a nylon membrane (also known as Southern Blotting).

7. Hybridization of the single strand of DNA with a radioactive probe which binds to specific sequences of DNA. (The final DNA fingerprint is built by using several probes simultaneously.)

8. Creating a picture of the radioactively marked DNA onto x-ray film.

After this is done you can visualize bands corresponding to the lengths of the fragments of DNA which can then be used to compare to another person’s for criminal trials or paternity tests.

PCR-based Analysis

1. Isolate DNA from blood, semen, or any other cell sample.

2. Extract the DNA from the cell.

3. Amplify the DNA by using the polymerase chain reaction (PCR). This DNA is copied over and over again usually in a machine called a thermal cycler.

4. Treat the amplified DNA with a variety of probes that are bound to a blot (probes on a membrane are dipped into the amplified DNA). Each probe is found in a specific “dot” on the blot strip. A chemical reaction causes the dot to darken and become noticeable when DNA containing a particle variant is present.

5. Infer the genotype by the pattern of dots that indicates which probes the amplified DNA bound to.

A short preparation and analysis time of a few days makes this DNA typing technique more efficient then RFLP which take a few weeks.

There are various other techniques for DNA typing. The technique will be determined depending on the situation and the amount of DNA available. All make use though of the tandemly repeated sequences present in a person’s DNA. There are some issues though with the accuracy of the tests and the possibility that the fingerprint may not be unique to one individual. These problems that arise and how they have affected the history of DNA fingerprinting will be examined.

The History of DNA Fingerprinting

I. Tiselius, 1933

a. Invented electrophoresis for separating proteins

II. Frederick Sanger, 1963

a. Developed sequencing procedure for proteins

III. P. H. O’Farrell, 1975

a. Invented two-dimensional electrophoresis

IV. E. M. Southern, 1975

a. Published his procedure for testing the existence of specific pieces of DNA

V. Alfred J. Jeffreys, 1984

a. Discovered the fundamental processes behind DNA fingerprinting

b. Studied gene for myoglobin

c. Found non-functional “minisatelites” – areas near gene which vary between individuals

d. When these segments were isolated, enhanced, and radioactively labeled they could be used to distinguish individuals through gel electrophoresis.

e. The original process could take up to six weeks, but by 1991 Jeffreys had improved the test so that it took as few as two days.

VI. 1985 – First paternity test

VII. 1988 – First criminal conviction based on DNA evidence

VIII. 1989 – First conviction overturned based on DNA evidence

a. Gary Dotson served 10 years of his 25-50 year sentence

IX. Kary Mullis, 1993

a. Won the Nobel Prize for the development of PCR (polymerase chain reaction) procedure.

b. Made isolation and analysis of DNA quicker and easier by reducing the amount of DNA needed from the organism.

c. He originally conceived the idea in 1983.

X. 1996 – First conviction based on mitochondrial DNA

XI. 1999 – First “cold hit” obtained from a DNA database

a. Wallid Haggag is convicted of burglary

b. He was not a suspect, but police matched blood found at the crime scene to Haggag’s blood which was on record in the state DNA database

Problems with DNA Fingerprinting

I. Costs – Time and Money

a. Can be very expensive and time-consuming

b. Less of a problem since Kary Mullis’s development of PCR

II. Chance

a. There are no clear matches – only probabilities.

i. The DNA fingerprinting process examines several pieces of DNA, not the whole sequence

I. Though unlikely, two individuals could have the same DNA fragments while being genetically distinct

II. Racial and ethnic groups are likely to have similar VTNR’s

III. The Human Factor

a. Comtamination

i. DNA from a lab technician or a police officer can become mixed with DNA from a suspect

b. The Jury

i. DNA fingerprinting can be confusing to explain to a jury.

ii. Any doubt about contamination can destroy the validity of DNA evidence

c. Misuse

i. Planting DNA evidence to frame a suspect

ii. Concerns about racial profiling stemming from similarities in VTNR’s

Applications of DNA fingerprinting/typing

1. Forensic analysis:

DNA typing can be used in forensic cases of murder, rape, homicide, and other violent crimes. DNA samples are typically taken during a criminal investigation, from the crime scene area or from the victims clothing or body and include: blood, hair, skin cells, or any other genetic material. The samples are then compared using VNTR patterns to determine a match either to the suspect or victim. VNTR patterns can also be used to identify victims of homicide when a sample from the parents is available. If the sample is extremely small PCR can be used to amplify the DNA for the typing tests.

2. Population genetics:

Used to determine variability in various ethnic groups or populations.

3. Providing pedigree status for certain animals:

Commonly used to determine specific breeds of some horses and dogs. This DNA fingerprint can be used for registering animals and establishing pedigree as well as for parentage verification. DNA identity information can be used to correlate EPDs to parent stock which allows selection of animals that meet set criteria for performance.

Benefits of Using DNA-based Animal Identity:

-Create a permanent identity record for each animal

-Increase value based on purebred or branded product verification

-Resolve pedigree disputes

-Confirm Parentage in multi-sire breeding programs

4. Forensic analysis of wildlife crimes:

Can be used to solve crimes against wildlife such as illegal hunting, trafficking in endangered species, and the production and sale of products made from illegally hunted animals. There is one lab in the world that deals with these cases, The National Fish and Wildlife Forensics Laboratory in Ashland, Oregon. They work on cases from the 50 United States and from 155 other countries worldwide. The lab staffs 33 people that work on approximately 900 cases every year. They have a reference warehouse that holds stuffed animals and animal parts used to identify creatures. They have nearly 5,000 complete animals and over 30,000 blood and tissue samples, mostly donated from zoos around the world after an animal dies. They use DNA in 15-20% of their cases to link animals and their human killers to a crime scene. In combination with ballistic evidence and fingerprints they have become very accurate at tracking illegal hunters and poachers. In one example a man returned home after an illegal hunt and washed and dried his clothes at home. The lab was able to use animal hairs found in the dryer’s lint holder to place him at the crime scene. They can also use barely visible bloodstains on clothing to track hunters down years later, and with absolute statistical certainty.

5. To study endangered animal species:

Helpful when trying to discover if an animal that has been found is really an endangered species or only a mutant of another species.

6. Detecting genetically modified organisms:

Used especially for agricultural identification. Many of the crops currently grown in the United States contain genes that were introduced in order to develop a new type of crop. They can be detected in the crop using PCR. Approximately 50-75% of produce and processed foods in grocery stores in the United States are genetically modified or contain genetically modified organisms.

7. Testing for e. coli and other illnesses carried by food sources:

Done with PCR, using primers specific to certain disease strains can test for pathogens like e. coli or mad cow disease in food such as hamburger meat.

8. Paternity and Maternity testing:

DNA paternity testing is the most accurate way, with 99.9% certainty, to determine paternity. It can be used for maternity testing also but that is quite uncommon because mothers usually give birth at a hospital or with others that can testify to her being the mother. (Example: maternity testing could be used on a child that was born at home and then anonymously left at an orphanage or hospital.) Such accurate testing is possible because a person inherits VNTR patterns from their parents, and every persons DNA is unique except for that in identical twins. VNTR patterns are extremely specific and can be accurately compared. A child will share one band with the biological mother and one with the biological father. They can be used for parent identification as well as biological parenthood in adoption cases.

9. Personal Identification:

This application of DNA fingerprinting has been discussed but is not in use at the current time. It would involve using the VNTR patterns of individuals as a type of genetic bar code to identify them. This is relatively impractical because it would be far too expensive and time consuming to analyze and store millions of VNTR patterns to be used as personal identification references.

These alligator snapping turtles were recovered in 2002 in Greenwood, Mississippi, in a sting operation that ended in the arrest of a seller.

Confiscated animal products, including tusks, tortoise shells, and stuffed birds, at the U.S. Fish and Wildlife National Forensics Lab.

Through the Grapevine
Photograph by Jim Richardson

Ringed by his handiwork, Cornell researcher Bruce Reisch examines the leaves of a Chardonnay grapevine for signs of fungal disease. Reisch and his colleagues are working to create Merlot and Chardonnay grapes that are genetically modified to resist diseases that now must be fought with fungicides. “Our goal is to come as close as possible to eliminating the need for spraying,” he says.

Building a Better Tomato
Photograph by Jim Richardson

A slide representing 20,000 tomato genes is projected onto Mark D’Ascenzo, a researcher at Boyce Thompson Institute for Plant Research in Ithaca, New York. Scientists there are trying to identify the genes that make certain tomatoes resistant to diseases. "We’ve isolated hundreds of genes that are interesting candidates," D’Ascenzo says, "but we’re still years away from understanding the whole picture." Once scientists do, the genes that are responsible for resistance can be synthesized and inserted into a new generation of tomato plants.

http://www.sumanasinc.com/webcontent/anisamples/dynamicillustrations/paternitytesting.html

Case Studies

Murder/Rape Case: State of Florida vs. Jones and Reesh

Murder at Rodman Dam, 1988

In July of 1987, teenagers Randall Scott Jones and Chris Reesh were target shooting with a 30/30 hunting rifle at the Rodman Dam recreation area in Florida. While they were shooting, Jones’ pickup truck became stuck in a sandpit. A fisherman suggested that the two ask for help from a nearby pickup truck. The two approached the truck where they found Kelly Lynn Perry and her fiancé, Matthew Brock, sleeping. Jones and Reesh debated on whether or not they should wake the two to ask for help.

The next morning, fishermen found the bodies of Perry and Brock in the woods next to the recreation area. Police investigation showed that each was shot in the head with a 30-caliber bullet. Brock was shot twice in the head, and Perry was shot once. It was also found that Perry had been sexually assaulted. The pickup was reported stolen.

In August, Jones was arrested in Mississippi driving Brock’s truck. Reesh was arrested the next day after Jones had said the two were together that night in July. Both were indicted on counts of first-degree murder and sexual battery.

A semen sample was taken from Perry’s body, and blood samples from Jones and Reesh were taken. They were compared in a laboratory that specializes in DNA fingerprint testing. The DNA fingerprint indicated that Jones had raped Perry.

Using DNA fingerprinting along with other evidence, events of the crime were put together. Jones confessed to shooting both Perry and Brock in the head at close range. The two teens then dragged the bodies into the woods. They towed Jones’ truck out of the sandpit with Brock’s truck, and the two drove off with both trucks. Later, Jones returned to the bodies, took them further into the woods, and raped Perry.

A representative from the DNA fingerprinting laboratory testified that the chance of another person having the same DNA fingerprint as Jones was one in 9,390,000,000.

The jury only deliberated for 30 minutes. The jury convicted Jones of two counts of murder, one count of burglary, one count of shooting into an occupied vehicle, and one count of sexual battery. The judge sentenced him to a double death sentence. This made the case the first in which DNA fingerprint evidence was used, and the death sentence was given. Reesh was sentenced to six years in prison and twenty years probation.

Human Paternity Case: Sarbah vs. Home Office

Ghana Immigration Case, 1985

The first practical test of DNA fingerprinting involved Christina Sarbah and the Home Office in England. Christina wanted to prove that Andrew, who had been living in Ghana with Christina’s estranged husband, was indeed her son.

Immigration officials held Andrew at Heathrow Airport, because they claimed that his passport was forged, or a substitution had been made. Member of Parliament Martin Stevens intervened and made it so the child could stay at his family’s home in London with siblings David, Joyce, and Diana.

The Hammersmith Law Centre, who provides legal aid to the underprivileged, put together large amounts of evidence, including pictures and statement by family members. Various tests to determine genetic characteristics showed that Christina and Andrew were almost certainly related. What the tests did not prove was whether or not Christina was the mother or merely an Aunt. This evidence was rejected at an immigration hearing, but deportation was put off pending an appeal.

Centre workers contacted Alec Jeffreys at Leicester University after they read in the local newspaper about a scientific discovery that could prove maternity, and they asked him to take on the case. Jeffreys accepted the case because he believed it would be an ideal test of the DNA fingerprint technology he had recently developed. He compared the DNA extracted form the blood samples from Christina, Andrew, the three other children, and an unrelated individual. The resulting DNA fingerprint would verify whether or not Andrew is Christina’s son.

It was questioned whether Christina was Andrew’s Aunt, a sister of his true mother. Andrew’s fingerprint contained about 25 bands that were inherited from his mother. The possibility that Christina is the true mother’s sister and yet happens to share 25 bands with her is about one in 600,000.

The case was complicated because of the lack of the father’s blood sample and the doubts about Andrew’s paternity. Jeffreys reconstructed the father’s fingerprint from bands present in the three undisputed children, but absent in Christina. About half of Andrew’s bands match bands in the father’s compilation, and the remaining bands were all present in Christina’s fingerprint. The possibility of this happening by chance is greater than one in a trillion.

Horse Paternity Case: Thoroughly Bred

Father or Son? 1989

Thoroughbred horses are bred primarily for galloping speed, which is based on a number inherited characteristics, including muscle mass, conformation, and cardiopulmonary capacity. These animals are carefully mated, and their ancestry can be traced back many generations. Verification of an animal’s parentage is key to its value.

This case involves a leading stud horse, from one of the most prominent thoroughbred lines, and his son. The father was retired from studding when low sperm count indicated he was becoming infertile. Mares without foal who had been mated to the father were then remated to the son. However, standard blood tests could not confirm whether father or son was the sire of the foals conceived during this changeover period.

The autoradiogram proved that the son sired the foal. Although the foal shared a band with the father, all of the foal’s bands are accounted for only by including two that originated with the son.

As a test of identity, DNA fingerprinting can positively determine thoroughbred parentage. However, DNA fingerprinting of horses is far more difficult than it is in humans. Due to centuries of meticulous inbreeding, all thoroughbreds are derived from a closed pool of genetic material. Even thoroughbreds not directly related share a common genetic heritage, and consequently, may also share a large number of DNA fingerprint bands.

Bibliography

Russell, P.J. (2002). iGenetics. San Francisco: Benjamin Cummings.

Sheindlin, Judge Gerald. Genetic Fingerprinting: The Law and Science of DNA. Bethel, CT: Rutledge Books, Inc., 1996.

On the Human Genome Project. http://homepage.smc.edu/hgp/history.htm#timeline

Basics of DNA Fingerprinting. http://www.biology.washington.edu/fingerprint/dnaintro.html

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MSN Encarta. http://encarta.msn.com/encyclopedia_761579857/DNA_Fingerprinting.html#endads

Kary Mullis’ Biography. http://www.karymullis.com/bio.html

Dolan DNA Learning Center: DNA Fingerprinting. http://www.dnalc.org/resources/aboutdnafingerprinting.html

DNA Fingerprinting in Human Health and Society. http://www.accessexcellence.org/AB/BA/DNA_Fingerprinting_Basics.html

How DNA Evidence Works. http://science.howstuffworks.com/dna-evidence3.htm

http://www.biotech.iastate.edu/biotech_info_series/bio7.html#anchor15180002

http://edition.cnn.com/2004/TECH/science/05/22/high.tech.poaching.ap/

http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml

http://www.geneseek.com/link.sp?page=ident-parent-cattle

http://www.lab.fws.gov/

http://www.biology.washington.edu/fingerprints/apps.html

http://www.nationalgeographic.com

http://www.dnalc.org/shockwave/dnadetective.html