What is DNA Fingerprinting? Each of us has the same chemical structure of DNA. But there are millions of differences in the DNA sequence of base pairs symbolized as A, T, C, G. This makes the uniqueness among us so that each of us except twins is different from each other genetically. DNA sequence thus can be just like a fingerprint to identify every person entirely. But there are so many base pairs in DNA sequences that it is hard and time-consuming to read out all the sequences. Scientists have developed many techniques in order to quicken the identification process. Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific. Every strand of DNA has pieces that contain genetic information which informs an organism's development (exons) and pieces that, apparently, supply no relevant genetic information at all (introns). Although the introns may seem useless, it has been found that they contain repeated sequences of base pairs. These sequences, called Variable Number Tandem Repeats (VNTRs), can contain anywhere from twenty to one hundred base pairs.

An example of Variable Number Tandem Repeats (VNTRs) on three chromosome pairs.

Every human being has some VNTRs. To determine if a person has a particular VNTR, a Southern Blot is performed, and then the Southern Blot is probed, with an RFLP probe through a hybridization reaction, with a radioactive version of the VNTR in question. The pattern which results from this process is what is often referred to as a DNA fingerprint. An RFLP probe is a labeled DNA PCR primer sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. Isolation of sufficient DNA for RFLP analysis is time-consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have.

Because VNTR patterns are inherited genetically, a given person's VNTR pattern is more or less unique. The more VNTR probes used to analyze a person's VNTR pattern, the more distinctive and individualized that pattern, or DNA fingerprint, will be. Criminal Identification and Forensics DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself. National DNA Databank: 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 August 2007, the database contained over 5 million DNA profiles in its Convicted Offender Index and about 188,000 DNA profiles collected from crime scenes but not connected to a particular offender. As more offender DNA samples are collected and law enforcement officers become better trained and equipped to collect DNA samples at crime scenes, the backlog of samples awaiting testing throughout the criminal justice system is increasing 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. CODIS is a computer software program that operates local, State, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. Every State in the Nation has a statutory provision for the establishment of a DNA database that allows for the collection of DNA profiles from offenders convicted of particular crimes. CODIS software enables State, local, and national law enforcement crime laboratories to compare DNA profiles electronically, thereby linking serial crimes to each other and identifying suspects by matching DNA profiles from crime scenes with profiles from convicted offenders. The success of CODIS is demonstrated by the thousands of matches that have linked serial cases to each other and cases that have been solved by matching crime scene evidence to known convicted offenders.

How to perform DNA Fingerprinting? DNA contains two strands which are joined together by the forces between the two nitrogenous bases on both strands. We can imagine that DNA is a long ladder with many rungs. Each rung contains a pair of nitrogenous bases which are distinguished into four different types: adenine, guanine, cytosine and thymine. They are in turn symbolized as A, G, C, T respectively. The sequence of rungs is different for different persons and it is just like paragraphs of alphabets. Most of the sequence can function to regulate protein synthesis and to regulate cell activities. The rest of the sequence, in fact, have no specific function but with high variability among people. So this variability can be used to identify each person. One common technology used in DNA fingerprinting is the RFLP (restriction fragment length polymorphism) analysis. The first step is to extract genomic DNA (entire DNA pool of a person) from a person. This can be done by using one's hairs or blood. The DNA molecules are enclosed in the nucleus of the cells, so the extract of cells needs to be lysed by breaking the cell membrane and the nucleus envelope to release the DNA molecules. The DNA molecules are now still packed in the form of chromatin by proteins. They are then treated with buffer, enzymes and chloroform so as to destroy the proteins. The pure DNA molecules can then be separated from the destroyed proteins by centrifugation.

The extracted DNA molecules are now in a very long chain and need to be cut into many fragments which are easier to be recognized. The DNA long chains are cut by restriction enzymes. A specific restriction enzyme can cut a specific locus on the DNA chain. For example, EcoR1 will only cut the chain at the sequence of GAATTC. The mixture of fragments is then allowed to run in the gel electrophoresis. The electrophoresis can separate the fragments by electrical charges and a pattern of the fragment can be obtained eventually. This pattern is different for different persons and hence can be used for distinguishing people. The DNA fragments are then transferred from the gel to a nylon membrane which is called a Southern Blot. The separated DNA molecules are now still colorless and so it is hard to see them for identification. Probes have to be added to make them visible on a photographic film. The probe is a small radioactive fragment of DNA which is complementary to and can match with the sequence of the DNA on the nylon membrane. This matching is called hybridization. The radiation from the probes can cause darkening on the photographic films. This results in the appearance of a pattern of dark bars on the film, which is the DNA fingerprint of a person.

Applications Forensic Uses Blood or fragments of tissues such as hair and skin cells may be left in the scene of crime. The DNA fingerprint of these cells from the scene can be revealed and are compared with the DNA fingerprint obtained from criminal suspects. If somebody's DNA fingerprint matches with that from the scene this will be a strong evidence showing that he has committed the crime. Also in the case of rapes, the semen left in the victims' vagina can also be extracted and be used to reveal the DNA sequence which is later compared with the criminal suspects' DNA fingerprints so as to find out who did the rape. Paternity A child can inherit most of the DNA fragment pattern from his parents. By comparing the DNA fingerprints between the child and the suspect parents, it is possible to identify who the parent of the child is.

Southern Blot A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin M. Southern. Other blotting methods (i.e., western blot, northern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Southern's name. As the technique was eponymously named, Southern blot should be capitalized, whereas northern and western blots should not. Method Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. The DNA fragments are then electrophoresed on an agarose gel to separate them by size. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.

The membrane is then exposed to high temperature (60 to 100 °C) (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently and covalently crosslink the DNA to the membrane. The membrane is then exposed to a hybridization probe. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon testes (sperm) DNA for blocking of the membrane . After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used. Result Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe. The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods.

The High Rise Killer DNA Fingerprint

Tampa, Fl - June 13, 2008 A maid, doing her daily cleaning at a posh condo, found Sarah Elizabeth Jones, 35, dead in her bathtub. The maid said that the doors were locked when she arrived. Ms. Jones, who worked for an import company in St. Petersburg, had been on vacation at the time her body was found. She was not known to have a boyfriend but one man had been by seen by neighbors entering her condo on at least two occasions. The victim appeared to have been killed after a struggle at her condo near the beach. Police found items strewn about the condo as well as blood in three different places including the bathroom where the body was found. The numerous areas of blood were analyzed by crime scene technicians and were found to have come from two different people. Police speculate that the killer was injured in the struggle with the victim leaving his or her blood at the condo. Some of the blood was matched to the victim, but other samples came from an individual other than the victim.

Police also found fingerprints belonging to at least three persons, including the victim. The fingerprints were sent to the Integrated Automated Fingerprint Identification System (IAFIS) run by the FBI. IAFIS returned a match on two different men. The first set of fingerprints belonged to Gerald Goss, 45, from New Jersey, who listed his occupation as librarian. The second set belonged to John Kennedy, 25, of Denver, who listed his occupation as writer. Search warrants were obtained for blood samples from each man. The samples were then compared using Restriction Fragment Length Polymorphism (RFLP) technology. The samples were run on an electrophoretic gel for comparison. From these samples police hoped to determine which man’s blood matched the blood found at the scene that would identify the killer. The DNA from four blood samples 1) the unknown blood found at the site, 2) the blood of the victim, 3) the blood of John Kennedy and 4) the blood of Gerald Goss were treated with the restriction enzyme HindIII and placed in an electrophoresis gel in lanes 2 thru 5 respectively. Lane 1 was used for the DNA length reference markers.

Teacher Southern Blot Instructions

Prior to the experiment the teacher needs to fill the “Gel Buffer”, “Fluor Tag” and “Wash” tubes with water. The “Fluor Tag” tube can be filled with water with a small amount (1-5 drops per gallon) of blue food coloring (VERY light blue) if you wish. --------------------------------------------------------------------------------------------------------------------- The Students Should Always Wear Gloves 1. Wet the sponge with water from the sink faucet.

2. Remove the nylon membrane from its plastic bag. Handle the nylon with forceps using the end with the label.

3. Place the sponge into the plastic tray.

4. With your fingers, carefully remove the gel from its plastic bag and place it on the sponge.

5. Place the wetted nylon membrane face-down onto the gel with the printed part at the bottom of the gel (the wells are at the top of the gel). Smooth out bubbles with a gloved finger.

6. Pour 50 milliliters of buffer (Gel Buffer) into the tray.

7. Place a two inch stack of paper towels on top of the membrane.

8. Let the DNA transfer proceed for 10-20 minutes.

9. Remove the paper towels.

10. Remove the nylon membrane with forceps using the end with the label.

11. Place the nylon membrane face up on a paper towel.

12. Put the gel back into its container and plastic bag.

13. Remove the sponge and empty the plastic tray.

14. Pour the tube of fluorescent probes (Fluor Tag) into the plastic tray.

15. Place the nylon membrane face down in the plastic tray.

16. Wait 5 minutes.

17. Remove the nylon membrane with forceps using the end with the label.

18. Place the nylon membrane face up on a paper towel.

19. Empty the plastic tray.

20. Pour the tube of wash buffer (Wash) into the plastic tray.

21. Place the nylon membrane face down in the plastic tray.

22. Wait 5 minutes.

23. Remove the nylon membrane with forceps using the end with the label.

24. Place the nylon membrane face up on a paper towel.

25. Examine the nylon membrane with a fluorescent light and fill in the Southern Blot data

Student Southern Blot Instructions Wear Gloves 1. Wet the sponge with water from the sink faucet.

2. Remove the nylon membrane from its plastic bag. Handle the nylon with forceps using the end with the label. 3. Place the sponge into the plastic tray. 4. With your fingers, carefully remove the gel from its plastic bag and place it on the sponge. 5. Place the wetted nylon membrane face-down onto the gel with the printed part at the bottom of the gel (the wells are at the top of the gel). Smooth out bubbles with a gloved finger. 6. Pour 50 milliliters of buffer (Gel Buffer) into the tray. 7. Place a two inch stack of paper towels on top of the membrane. 8. Let the DNA transfer proceed for 10-20 minutes. 9. Remove the paper towels. 10. Remove the nylon membrane with forceps using the end with the label. 11. Place the nylon membrane face up on a paper towel. 12. Put the gel back into its container and plastic bag. 13. Remove the sponge and empty the plastic tray. 14. Pour the tube of fluorescent probes (Fluor Tag) into the plastic tray. 15. Place the nylon membrane face down in the plastic tray. 16. Wait 5 minutes. 17. Remove the nylon membrane with forceps using the end with the label. 18. Place the nylon membrane face up on a paper towel. 19. Empty the plastic tray. 20. Pour the tube of wash buffer (Wash) into the plastic tray. 21. Place the nylon membrane face down in the plastic tray. 22. Wait 5 minutes. 23. Remove the nylon membrane with forceps using the end with the label. 24. Place the nylon membrane face up on a paper towel. 25. Examine the nylon membrane with a fluorescent light and fill in the Southern Blot data sheet.

b

Teacher Key “The High Rise Killer”

115 bp

109 bp

103 bp

95 bp

84 bp

75 bp

62 bp

54 bp

43 bp

30 bp

1 2 3 4 5

DNA Marker Unknown Victim Suspect #1 Suspect #2

b

Student Worksheet “The High Rise Killer”

115 bp

109 bp

103 bp

95 bp

84 bp

75 bp

62 bp

54 bp

43 bp

30 bp

1 2 3 4 5

DNA Marker Unknown Victim Suspect #1 Suspect #2

b