DNA Fingerprinting

From Chempedia

The Processes of DNA Fingerprinting

DNA Fingerprinting – Introduction DNA, or Deoxyribonucleic acid, is a chemical structure that forms chromosomes which differentiate people’s individual traits. The structural build of a DNA molecule is a double helix which consists of two strands of genetic material spiraling around one another. The strands of DNA vary by having different orders of base pairs. There are millions of base pairs in each individual strand of DNA, so using these sequences would be time consuming. Instead, scientists use the repeating pattern found on DNA to determine whether two DNA samples are from the same person and of if the donors are related. The process of checking DNA sequences and mapping them onto an individual’s unique strand arrangement is called DNA Fingerprinting (Meeker-O’Connell).

DNA Fingerprinting– Isolation of DNA The first step in the DNA fingerprinting process is performed by collecting a substance (blood, semen, saliva, etc) from which the DNA would be obtained by cutting, swabbing, or scraping the substrate from the sample. For example, a blood-stained fabric is placed into a tube with an extraction buffer. The tube is gently shaken so that the biological material from the substrate comes off. A buccal swab, used to extract cheek cells, is usually utilized when obtaining a DNA sample from a live person (Kobilinsky). The vast majority of DNA is in the nucleus of a cell. Other particles such as proteins, RNA, lipids, other organic and inorganic molecules also exist in the sample taken. The objective of the isolation is to acquire high molecular weight, more than 20 kilobases (kb), of DNA. There are many techniques used to extract DNA. One is the organic extraction of liquid whole blood. Since there is no DNA in erythrocytes, red blood cells, they are just broken down into smaller forms by rupturing their membranes. Substances are added to digest proteins cell membranes, leaving DNA in its aqueous form. The Microcon 100 is used to filter out smaller broken down molecules, leaving us with the desired purified double-stranded DNA (Kobilinsky). The purified DNA is then quantified to make sure that there will be enough ’copies’ available for the successful testing. Many procedures such as ultraviolet and visible spectrophotometry, fluorometry, capillary electrophoresis, and yield gel electrophoresis may be used to do this (Kobilinsky). The most common is yield gel electrophoresis, or random amplification, where a short and arbitrary primer is used on the DNA fragments for amplification (Lovell). Another method used to reveal the fingerprint is by forcing the DNA strand to copy itself using a process called Polymerase Chain Reaction (PCR). This method is useful, as it does not require the use of a living organism, such as yeast, to carry the DNA strand and perform cell division (Housman). When applied, the PCR method is able to construct allelic profiles by isolating short oligonucleotide regions, or RNA sequence, to be combined with the target species. This new chain is then manipulated into an original DNA Polymerase by repeatedly altering the temperature. The product of this amplified reaction usually is created as a band suspended in a gel. When multiple genes are manipulated in this way the procedure is known as a multiplex Polymerase chain reaction which increases the speed of testing (Nickell).

DNA Fingerprinting Process – Cutting, Sizing, and Sorting Enzymes are organic catalysts that speed up chemical reaction rates, and like all proteins, each one works best at a certain optimum temperature and pH. They exist in animal and plant cells, as well as viruses and bacteria. When alien genetic materials manage to enter bacteria, their restriction enzymes destroy the foreign DNA. In laboratories, over 100 of these enzymes have been extracted and studied, and we know that they work on different restriction sites by varied base pair sequences. The enzyme responsible for cutting the DNA sample into fragments is called the type II restriction enzyme, which acts on specific areas called restriction sites (Koblinsky). The restriction enzymes cut DNA strands by breaking the sugar-phosphate bond located between the nucleotide bases, thus identifying the short base sequences. An example is the Hae III, which recognizes the sequence 5’-GGCC-3’ and cuts between the G and C bases (Kobilinsky). In the human genome, there are about 3.1 million different subunit sequences that can vary from person to person-except in identical twins and clones. For this reason, each person has specific proportions of different sized fragments. Gel electrophoresis is used to separate these fragments and facilitate their sorting based on their masses (fragment sizes). In preparation of the gel plate, agarose or acrylamide gel mixture is poured onto a glass plate, producing a thin gel layer. A comb is inserted at one end of the plate for easy removal of the gel after it hardens, and will present small wells within the gel. Because DNA has a net negative charge in a neutral solution, the negative electrode, the cathode, is placed on the end of the plate with wells. The anode, the positive electrode, is then placed at the opposite end (Koblinsky). 50-100 nanograms (ng) of purified DNA are then placed into each of the small wells via the pipette. When the electrodes are turned on, there is a voltage difference across the gel, and the negatively charged DNA moves in the gel away from the cathode toward the anode. To speed up this process, higher voltage difference may be applied. The DNA fragments are separated by size because the larger the fragment is, the slower it will move in the gel. On the contrary, the smaller and lighter fragments move faster and travel a greater distance in the gel (Kobilinsky).

DNA Fingerprinting Process – Data Analysis After the DNA fragments are cut, a process called Southern Blotting (see Figure) is used to transfer them onto a nylon sheet. This procedure enhances, or detects, abnormalities in the DNA gene sequence. DNA with high molecular weight is isolated from the selected strand by gel electrophoresis. In this pure form it can be transferred as a gel onto a nylon membrane. The membrane has been soaked in a solution containing a radioactive probe (Lovell). Geneticists are able to identify targets using autoradiography, or a colorimetric reaction (Nickell). This process is also useful in detecting mutations for the diagnosis of viral diseases, cancers, and even identifying X and Y chromosomes to identify the gender of the individual (Housman).

Figure I: Southern Blotting Technique- here the abnormalities are detected through electrophoresis by transferring the material onto a nylon membrane in gel form.

(Meeker-O’Connell)

Once in gel suspension, a radioactive probe narrows in on a target by using the radiation-bound DNA structures, or allele sequences, on the nylon membrane (Wikipedia). Under an X-ray, the radioactive pattern forms a visible band-like pattern that is as unique as the DNA itself is to the individual. This is the DNA "fingerprint" (Lovell). Once the radioactive probe is stuck to its target on the membrane, a picture may be taken using a camera and a specialized film which absorbs the photon light energy. Data can also be retrieved from the radioactive probe using an x-ray film to see the repeated sequence of the locus, called the variable number of tandem repeats (VNTR), of the DNA. For accuracy, four to five different radioactive probes are used on different parts of the VNTR sequence. Using four probes will give eight pieces of information about an individual (Meeker-O’Connell).

The Importance of DNA Fingerprinting- Real-life uses A main reason for the utilization of DNA fingerprinting is to assist in the criminal and social justice system. It may be used to identify the deceased person, validate a suspect, confirm a rape, and identify the host of a blood splatter (Noguchi). DNA evidence is highly accepted and often used in trials and other justice systems. Another application to DNA fingerprinting is to diagnose inherited or genetic disorders in both adults and children such as Alzheimer’s, sickle cell anemia, cystic fibrosis, and Tay Sachs disease. Geneticists hope that by perfection the identification of these disorders a solution may be more readily found. DNA fingerprinting can also be applied to paternity tests to be used by both the parent and child to identify the offspring’s father. In conclusion, when one thinks of DNA fingerprinting and the biological processes associated with it, it becomes difficult to identify the chemical steps necessary to perform such a complex task. Yet, when considered at the molecular level, the building blocks of DNA and the fingerprinting process have very much to do with chemical reactions that take place inside of each organic particle. Thus, without chemistry, geneticists would never be able to fully comprehend the complex workings of even the tiniest biological forms, such as DNA, which would never fully take form without the basis of chemical exploration.

Works Cited

1. Genetic Fingerprinting. 20 Sept. 2005. Wikipedia. 27 Sept. 2005 <http://en.wikipedia.org/wiki/Genetic_fingerprinting>.

2. Housman, David E. "DNA on Trial - The Molecular Basis of DNA Fingerprinting." The New England Journal of Medicine. 23 Feb. 1995. 332:534-535. 17 Oct. 2005 <http://content.nejm.org/cgi/content/full/332/8/534>.

3. Kobilinsky, Lawrence, Thomas F. Liotti, and Jamel Oeser-Sweat. DNA: Forensic and Legal Applications. Hoboken, New Jersey: John Wiley & Sons, Inc., 2005.

4. Lovell, Mark A. Molecular pathology. 30 May, 2002. Access Science (McGraw-Hill).

5. 8 Nov. 2005 <http://www.accessscience.com.floyd.lib.umn.edu/>.

6. Meeker-O’Connell, Ann. How DNA Evidence Works. Howstuffworks. 19 Sept. 2005 <http://science.howstuffworks.com/dna-evidence2.htm>.

7. Nickell, Joe, John F. Fischer. Crime science : Methods of Forensic Detection. Lexington: University Press of Kentucky, 1999.

8. Noguchi, Thomas T. "Forensic medicine". 2002 AccessScience (McGraw-Hill). <http://www.accessscience.com.floyd.lib.umn.edu, DOI 10.1036/1097- 8542.268500>

Footnotes

Researched and written by: Shalene Sankhagowit, Erin Weiland, and Thao Tran