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DNA Fingerprinting

DNA Fingerprinting

Goals Your active participation in these laboratory exercises will: Enable you to understand methods of human identification Introduce you to new terminology and concepts Expose you to common techniques used in biotechnology Teach you how to perform an agarose gel electrophoresis Introduce you to non-coding DNA (“junk DNA”), its variability, importance and potential uses • Enable you to be an “investigator in a CSI lab” and “solve a crime” • • • • •

Introduction DNA fingerprinting is a technique used to distinguish between individuals of the same species using DNA polymorphisms. Any two humans will have the great majority of their DNA in common. DNA fingerprinting analyses highly repetitive sequences called minisatellites that vary from person to person. One way to perform DNA fingerprinting is by RFLP analysis (Restriction Fragment Length Polymorphism). The DNA containing the variable amounts of repeated sequences is subjected to restriction endonuclease digestion in which the DNA is cut into fragments by specific restriction enzymes. The restriction fragments are then separated according to their sizes on agarose gels. The DNA is visualized by Southern blotting, or by staining the agarose gel after the electrophoresis. If PCR (Polymerase Chain Reaction) is used to amplify the DNA, fluorescent PCR primers may be used in the polymerization step. Subsequently, the migration of the amplified DNA fragments through the gel can be visualized by fluorescence detectors. Background Although fingerprints have been used for personal identification since ancient times, they have only been used in criminal cases since the 20th century. The term “DNA fingerprinting” is used to describe an identification technique that analyzes genotypic information rather than relying on the phenotypic information obtained from traditional fingerprints. Another type of phenotypic identification can be obtained through blood typing and leukocyte antigen testing. However, this type of identification can only be used to exclude certain suspects rather than identifying them. DNA fingerprinting, on the © B. Woelker, Ph.D., 2008

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other hand, can provide positive identification of a suspect since no two individuals (with the exception of identical twins) will have the same DNA fingerprints. In 1987, Florida rapist Tommie Lee Andrews was the first person in the United States to be convicted as a result of DNA evidence, for raping a woman during a burglary. DNA fingerprinting has been used to: 1) identify the guilty and the innocent in murders and rapes, 2) to confirm parental identity, and 3) evolutionary studies. British researcher Alec Jeffreys was the first to analyze DNA polymorphisms to be used for human identification. In 1984, Jeffreys discovered highly repetitive DNA sequences (called minisatellites) ranging in size from 9-80 bp in the human genome. The repetitive DNA is localized in non-coding regions, also called “junk DNA”. Large amounts of repetitive DNA are found near the centromeres and at the telomeres. One reason for repetitive sequences at the telomeres is marking the age of a cell. Others are their importance in recombination events and gene expression, but in general, very little is known about the function of the vast amounts of repetitive DNA sequences in human DNA. Jeffreys found that the number of repeats in a particular locus was variable between homologous chromosomes and more importantly between individuals. He named them VNTRs (Variable Number Tandem Repeats) and STRs (Short Tandem Repeats) depending on their sizes, respectively. Restriction Enzymes Restriction enzymes recognize short double-stranded sequences of DNA and cut both strands of the molecule between the sugar and the phosphate in the sugar-phosphate backbone. Restriction enzymes are bacterial enzymes than protect the bacteria from phage infection. Today, more than 300 different restriction enzymes are known; most of them are purified and available for uses in biotechnology. Gel Electrophoresis and Analysis Agarose gel electrophoresis uses agarose, a polysaccharide purified from a seaweed named Agar agar as a matrix. Agarose solidifies at 45ºC. It will make a molecular sieve with a meshwork that is dependent on the agarose concentration. Typically, 0.8% agarose gels are used which will separate DNA fragments between 800 and 50,000 base pairs. 1% or 2% gels are used for smaller DNA fragments. Migration through the gel occurs in an electric field. DNA is an acid: it can donate hydrogen ions from its phosphate groups and thus is negatively charged. Its separation is therefore mainly dependent on its molecular weight. If DNA is subjected to an electrical field, it will migrate from the cathode (negative pole) to the anode (positive pole). A buffer, which is an aqueous solution that contains salt ions and stabilizes the pH, is used to conduct electricity through the gel. TBE buffer is a combination of Tris, Boric Acid and EDTA. DNA is usually colorless. To monitor the migration through the gel, the samples are mixed with loading dye. The pigment in the loading dye, bromophenol blue (purple) mimics linear double stranded (ds) DNA fragments of about 300 bp length. A second loading dye, xylene cyanol (light blue), that mimics longer dsDNA fragments of about 4000 bp length, may be added as well. The loading dye serves a second function. It contains ficoll, a dense polysaccharide that allows the sample sink into the well of the agarose gel. The loading dyes will indicate where to expect the DNA during the gel electrophoresis.

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DNA Fingerprinting

The DNA still has to be visualized. This is done by either Southern blotting or posttreating the agarose gel with DNA stains like methylene blue (Carolina Blu®) or ethidium bromide. In the last step, the pattern of DNA fragments is compared to the restriction fragment pattern of a known sample, e.g. the evidence DNA at a crime scene. At crime scenes, the amount of usable DNA found is usually small, hence a PCR amplification step prior to the separation of the fragments might be necessary. If PCR is used, one strand of DNA can be amplified 1 billion times in 30 cycles (230). The desired fragment will have the chosen PCR primers as the boundaries and thus the restriction digest step might be unnecessary. The FBI currently uses 13 different DNA regions for human identification. If used properly, DNA fingerprinting is a very powerful tool in human identification. Besides nuclear DNA, mitochondrial (mt) DNA is also used in crime investigation, genealogy and evolutionary studies. For the crime scene investigation, mtDNA is of great importance, since many older samples lack useable nuclear DNA. mtDNA is inherited through the maternal line only. A hypothetical fingerprinting is illustrated below: The crime scene evidence came from blood on the victim’s clothes. Three suspect DNA samples are compared to the evidence DNA.

Repeating dinucleotide (AT)100

Repeating dinucleotide (AT)100

Repeating dinucleotide (AT)300

Restriction sites

1000

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Restriction enzyme cuts DNA into small fragments

1.

3100

1000

2. E.

3. 1.

2.

3.

3100 2300 2100

1000

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The DNA fragments are separated through gel electrophoresis and visualized. The evidence DNA (E) is compared to the suspects DNA (1, 2 and 3).

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DNA Fingerprinting

Crime Scene Investigation (CSI) Scenario I: A murder has happened. The CSI team collects blood from the crime scene to find the murderer. Three possible suspects (S1-the neighbor, S2-a colleague of the victim and S3the wife) are investigated. Use Evidence A (EA) DNA from the crime scene blood. Scenario II: Bill is incarcerated since 1983 for a murder and rape he says he did not commit. His defense layer asks to reopen the case and analyze a probe of semen found on the victim at the time of the crime. Use DNA from semen (EB) to see whether Bill (S1) is innocent. Other suspects may be Rob (the former husband –S2) and Anthony (the high school boyfriend –S3). Scenario III A young mother demands alimony from a man who she claims is the father of her newborn child. The man refuses. He claims the baby must be from another man. DNA testing is performed. Similarity among the RFLP bands could point to the most likely father. EC is a sample of DNA taken from the baby. S1 is DNA taken from the accused man, two other samples (S2) and (S3) are taken from the mother’s former boyfriend and the gardener, respectively.

Part A: Gel electrophoresis Materials

Per Class 0.8% agarose, kept warm in Styrofoam box Carolina Blu® stain (methylene blue) Sharpie® markers or wax pencils Per Table Group 1 electrophoresis chamber 1 power pack 1 gel casting tray and comb electrophoresis buffer (TBE) 1 eppendorf micropipette (0.5- 10µl) and tips Eppendorf tubes containing DNA samples (EA, EB, or EC, S1, S2 and S3), mixed with loading dye 1 container with lid for gel staining Transilluminator or light box

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DNA Fingerprinting

Procedure

1. Obtain all needed materials for this laboratory activity. 2. Review the procedures for this laboratory activity with your lab partner(s). Ask clarifying questions of one another and/or your instructor before doing anything if you don’t understand the procedures. 3. As you perform this procedure keep careful records of your results in the appropriate section of this laboratory exercise. Obey all lab safety rules. 4. Insert the comb into the casting tray. Use the melted agarose to pour a gel as shown by your instructor. 5. After about 10 min, the gel is solidified. Lower the barriers on both sides of the casting tray and insert it into the electrophoresis chamber. Make sure, you insert the tray in the proper orientation. Remember: DNA is negatively charged. 6. Fill the chamber with TBE buffer until the buffer just covers the gel. 7. Remove the comb carefully. 8. Load 10µl of your evidence sample (EA, EB or EC) into the second well from the left. Leave one well empty and using a different pipette tip each time, load your three suspect samples (S1, S2 and S3). 9. Once the wells have been filled, put on lid, connect electrodes and apply power (130 Volts) to the chamber. Turn the power off when the bromophenol blue dye has migrated three quarters through the gel (after about 30 – 45 min.) 10. Stain the gel using methylene blue stain (Carolina Blu®) for 10 minutes. Recycle the stain and de-stain the gel with deionized water for 30 minutes to overnight. 11. Analyze the gel using a transilluminator and record your data.

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DNA Fingerprinting

Results

Record your observations and results for procedure A as indicated. Label the diagram below. _

+ _ _ _ _

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Name(s): ____________________________________________ Section: _____________ Date: _____________________ General Note: Your instructor may require you to hand this portion of your laboratory in as a report of your work. Print neatly using a pencil for data and a blue or black ink pen for the questions. Make sure the complete name(s) of all members of the group are clearly given above.

Analysis:

With the members of your group, complete the data analysis for this laboratory as indicated below. Compare your DNA in lane 2 (EA, EB or EC,) with your suspect DNA (S1, S2 and S3). Identify a match by comparing the patterns of DNA bands on the Gel. Who may have been the perpetrator of the crime (or father of the baby)?

Conclusions:

To complete your work for this laboratory, answer the questions below in the space provided. 1.

Did you accomplish your goals? Explain in detail how you accomplished each of the goals outlined above.

2.

How did specific aspects of the procedures performed in this laboratory support your goals and/or reinforce the introductory information given at the beginning of this chapter? Explain in detail.

© B. Woelker, Ph.D., 2008

DNA Fingerprinting

3.

Why are the DNA fragments forming “bands” in the gel?

4.

Which of the DNA fragments in the gel are longer, the ones near the top or the ones near the bottom? Explain.

5.

Why can you observe bubbles on the cathode when power is applied to the electrophoresis apparatus?

6.

Why does the restriction digest create different patterns of bands for different individuals?

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What are sources of possible errors in DNA fingerprinting?

8.

List some properties of “junk DNA” in the chromatin.

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