Deoxyribonucleic acid (DNA) is contained in every cell of the human body.  It is the "blueprint" for life. Certain analytical techniques have been developed that are very useful in court cases today. The chemical structure of the molecule is important to understand when utilizing these techniques if any sort of intelligible results are to be obtained. DNA evidence has a very strong bearing on many cases across the world.

DNA is composed of a series of nucleotide bases. The bases consist of a nitrogenous group, a pentose (five-carbon) sugar, and a phosphate group. The sugar and the phosphate combine to form a "backbone" for DNA. Phosphate "is an anion formed by [breaking down] the neutral acid phosphoric acid (H₃PO₄)." When the acid breaks down, it loses the three hydrogen atoms. The loss of the three positively charged hydrogen atoms creates a phosphate group (PO₄^3-). Since the acid originally had a neutral charge, the loss of three positive charges drops the overall charge of the phosphate group to negative three (Campbell 61). This gives DNA an overall negative charge, which is useful for analytical techniques. The phosphate group is an essential part of the nucleotide base.

DNA is "double stranded with the sugar phosphate backbone on the outside of the helix" (Campbell 88). The backbone is on the outside of the helix for a good reason: the backbone is hydrophilic. That is, it is attracted to aqueous (water based) solutions, while the internal nitrogenous groups are hydrophobic, meaning they aren’t attracted to aqueous solutions. Since DNA is found in the cell and cellular fluid is aqueous the hydrophilic parts of the molecule will naturally form the characteristic external double helix of DNA.

There are two families of nucleotide bases; purines and pyrimidines. These two families differ only in their ring structures. The ring structure is literally a ring of carbon and nitrogen atoms that make up the nitrogenous group of the nucleotide (Campbell 84). Furthermore, there are two types of purines and two types of pyrimidines. Adenine (A) and Guanine (G) are purines while Thymine (T) and Cytosine (C) are pyrimidines. The bases are stacked on top of each other and form the two strands of the DNA helix. However, pyrimidines are always paired with purines. In 1953, Watson and Crick discovered that "the percentage of adenine…usually equaled the percentage of thymine. Similarly, the percentage of guanine equaled the percentage of cytosine" (Watson and Crick). From this information they concluded that A always pairs with T and C always pairs with G. The pairing of the molecules is known as base pairing (Campbell 85). The molecules bind because of their structures and hydrogen bonding.

Hydrogen bonds are weak bonds that form between the slightly positive hydrogen of one molecule to the slightly negative oxygen of another molecule. A single hydrogen bond is very weak, but the series of hydrogen bonds down the internal structure of the molecule, between the bases, acts like a zipper that helps DNA to hold its shape (Campbell 88). The series of purine and pyrimidine bases bonded together with hydrogen bonds is characteristic of the DNA molecule.

The two strands of a DNA molecule are said to be complimentary because the bases compliment each other all the way down the strand. This is one of the most important properties to know when utilizing certain analytical techniques (Campbell 306). Since the base pairs of DNA can theoretically be arranged in any order, the number of sequence possibilities on any given strand of DNA is virtually infinite (Campbell 306). This, coupled with the fact that "in humans [DNA] can be as much as 12 centimeters long and have a mass of 150 billion hydrogen atoms" illustrates that DNA varies greatly from person to person (DNA Structure). It varies so greatly that DNA is "person-specific", that is, each person has DNA unique to him or her. However, while "most sections of [DNA] are largely the same among humans, certain segments are variable." These varying segments are known as polymorphisms (Illinois v Kerry L. Pope). It is because of these polymorphisms that "no two individuals (except for identical twins) have identical base sequences throughout their DNA" (Illinois v Kerry L. Pope).

      Model of DNA showing base pairing and the sugar-phosphate backbone
 

There are many techniques to analyze DNA and they all rely on the structure of the molecule in some way. Three main techniques are used today. They are polymerase chain reaction (PCR)/reverse dot blot analysis, gel electrophoresis, and DNA fingerprinting, which relies heavily on restriction fragment polymorphism (RFLP) analysis. All of the techniques analyze DNA from a given source. Almost all biological evidence can be subjected to DNA tests. This includes hair, urine, skin, and blood, which is found at 60% of murders, assaults, and battery crime scenes (Weedn 2).

PCR is "a technique by which any piece of DNA can be quickly amplified in vitro" (Campbell 399). This technique is used when there is little DNA available to test or if the DNA has been degraded in some way. The PCR technique alone is not used to analyze DNA. It is usually coupled with other techniques, such as the reverse dot blot technique (Weedn 7). The procedure is begun by gently heating the sample of DNA. This process excites the molecules and breaks the hydrogen bonds between the bases of the two strands. Now the strands have been separated and the procedure can continue.

The newly separated DNA is incubated with the enzyme DNA polymerase, free nucleotides and other short pieces of DNA called primers (Campbell 399). DNA polymerase is found naturally in the cells of the body. It is the enzyme that kick starts DNA self-replication.

Once the enzyme reacts with the DNA, a polymerase chain reaction starts. The primers play just as important a role in the chain reaction as the polymerase does. They are there to prime, with a chemical signal, certain DNA sequences to accept other free nucleotides that are in the solution. Once the DNA has been separated and primed, the replication begins. The nucleotides begin to bond, via hydrogen bonding, to the open DNA strands and create two new identical strands of DNA. The process is repeated about forty times to create billions of copies of the target DNA sequence (Campbell 40). The amplified DNA can now be used in further testing to find some conclusive evidence.

The just-amplified DNA is normally used in a reverse dot blot analysis. This relies on the theory that in any given region of DNA there is a finite possibility of sequences.  Probes are used to see which of these sequences are present (Weedn 7). A common form of this PCR/reverse dot blot procedure is the DQ-Alpha procedure. In this form of analysis "the portion of DNA to be identified must be part of a well-characterized gene and must be polymorphic" (Forensic uses of biotechnology). It is a procedure in which "an analyst looks at one particular gene, the DQ-Alpha gene, which is…inherited and appears in varying forms in different people." This procedure is very popular because the target sequence is polymorphic and the gene is relatively easy to isolate. Once the PCR has taken place, "the amplified DNA is flooded over a nylon membrane onto which have been dotted a number of…probes, each of which is designed to recognize one [possible] sequence on the targeted gene" (Illinois v Kerry L. Pope).

These "probes" are short strands of DNA with specific sequences. They will bind only to complimentary strands of DNA in the given sample. For example, if the probe has the sequence ATGCTTATGCGCGAA, then it will bind only to a strand of target DNA with the sequence TACGAATACGCGCTT. A blue dot appears on the nylon membrane anywhere a probe has found a complimentary strand of DNA to bind to. The application of this technique is quite simple. The sample DNA and DNA from a suspect is subjected to identical procedures. If the two membrane patterns match each other, then the suspect is included in the pool of possibilities. If there is no match, he or she is completely ruled out. This test is useful for excluding suspects only. The reason is that there are only so many possibilities for sequencing in any given DNA sequence, and it is perfectly plausible that two people have the same sequence in the same area. In the case of the DQ-Alpha gene, there are only 21 possibilities for sequencing of the base pairs (Illinois v Kerry L. Pope). More conclusive evidence requires the use of restriction enzymes.

A restriction enzyme is an enzyme (or endonuclease, because it usually found in the nuclei of cells) that cuts DNA at a specific base sequence, which is usually approximately four to eight base pairs long. For example, the restriction enzyme EcoRI cuts DNA at the sequence G*AATTC / CTTAA*G (Restriction Enzymes – Background). The asterisks represent the sites of the cut. This results in two pieces of DNA that have staggered ends (Uitterlinden and Vijg 7). The sequences AATTC and CTTAA are now unbound. The now unbound nucleotides created are ready for other nucleotides to come and bind to them. They are "sticky". Therefore these restriction fragments (small fragments of DNA that have been cut by restriction enzymes) are said to have "sticky ends". In nature, they are found in the nuclei of certain bacteria. The bacteria use them to cleave foreign DNA that could be very harmful to their survival. Scientists have put these enzymes to good use, and they can now be manufactured in laboratories (Campbell 391). Once DNA has been cloven by restriction enzymes, it can be used in other laboratory techniques, such as gel electrophoresis.

Gel electrophoresis separates macromolecules (DNA, RNA or proteins) on the basis of size, electrical charge, or other physical properties (Campbell 393). This procedure is used most often in criminal cases. DNA is cloven by restriction enzymes
and placed in "wells" in a thin gelatinous membrane. The gel is supported by glass plates, placed in a chamber, and bathed in an aqueous solution. Electrodes are connected to the chamber and an electrical current is run through the solution (Campbell 396). The chamber has a positively charged end and a negatively charged end. Since DNA has an overall negative charge, due to the presence of the phosphate groups, it migrates towards the positive end of the chamber when influenced by the electrical current.

The size of the restriction fragments varies. It depends on where the enzymes used cut the DNA, and how long the sequences between each cut are. A 20 base pair sequence of DNA is going to weigh considerably less than a 10,000 base pair sequence. In general, smaller molecules move through the media with greater ease, because they can fit through pores easier than the larger molecules can. Similarly, molecules with a high negative charge move through the media more quickly than molecules with a low negative charge. All the molecules move through the gel at the same "speed" because they all have similar size to charge ratios (Martin 13). However, their sizes cause them to "bump" through the media and they separate into bands. The bands become visible on the gel only after staining (Campbell 396).

A more specified form of gel electrophoresis is known as restriction fragment length polymorphism (RFLP) analysis. It is based on naturally occurring polymorphisms in the human genome (Campbell 403). As discussed earlier, polymorphisms are various sequences in human DNA that vary from person to person. The starting material in general gel electrophoresis is the entire DNA strand from the source material. Since human DNA is so long, the bands on the gel would not be distinguishable if all of them were stained. The DNA would appear as one large smudge instead of a series of small, distinct bands (Campbell 403). To solve this problem, scientists manufactured slightly radioactive probes that bind only to specific segments of DNA, depending on the base sequence. These segments are polymorphic. This probe is poured over the gel, and a film is placed over the gel. The radioactive probes react with the film to make a "picture" on it that can be viewed under certain types of light. The result is a few distinct bands instead of a large smudge (Campbell 403).

RFLP analysis provides courts with a DNA "fingerprint". A DNA fingerprint is a set of RFLP markers used to identify a specific person’s DNA (Campbell 409). The application of this technique is extremely useful in criminal cases. A suspect's DNA is subjected to the same procedure as crime-scene DNA. If the two sets of bands match up, then the person can be identified as the guilty party with almost 100% certainty. If they do not match, then the person can be removed from the list of suspects. This is much more reliable than the DQ Alpha testing because of the specificity of RFLPs in human DNA.

Much controversy surrounds DNA forensics and biotechnology. There is an ongoing debate over whether or not DNA fingerprints should be kept on file, or destroyed after their use in a case. Others argue that DNA forensics is too immature a science to be considered admissible in any case (Weedn 2). However, many states are already deeming it admissible. In fact, in 1997, "more than 17,000 cases involved forensic DNA evidence in the USA alone" (Weedn 1).

DNA analytical techniques are extremely useful. Not only can they be used to convict criminals or exonerate the innocent, but analyzing DNA can lead to cures for genetic disorders that plague society today (Campbell 398). Without knowledge of the structure of the molecule, none of this would be possible. DNA structure is essential to know and understand in order to successfully apply analytical techniques.
 
 

1.  Campbell, Neil A.  Biology, Third Edition. Redwood City, California: The Benjamin/Cummings Co, 1993.

2.  Cook, P.J., and Green, J.  "Illinois v Kerry L.Pope."  Online Posting.  30 April 2000.
              http://www.state.il.us/court/appellates/4950021.txt

3.  "DNA Structure." Online Posting. 30 April 2000. http://www.chem.wsu.edu/Chem102/102-DNAStruct.html

4.  "Forensic Uses of Biotechnology." Online Posting. 30 April 2000. http://ntri.tamuk.edu/cell/pcr/pcr.html

5.  Martin, Robin. Gel Electrophoresis: Nucleic Acids. Oxford, UK: BIOS Scientific Publishers, 1996.

6.  Peters, Pamela. "Restriction Enzymes - Background." Online Posting. 30 April 2000.
              http://www.accessexcellence.org/AE/AEC/CC/restriction.html

7.  Peters, Pamela. "The Structure of DNA Molecule." Online Posting. 30 April 2000.
              http://www.accessexcellence.org/AE/AEC/CC/DNA_structure.html

8.  Uitterlinden, Adnre, and Vijg, Jan. Two Dimensional DNA Typing: A Parallel Approach to Genome Analysis. New York:
                E. Horwood, 1994.

9.  Weedn, Victor. "The Unrealized Potential of DNA Testing." National Institute of Science - Research in Action. Washington,
                D.C.: US Department of Justice, Office of Justice Programs, National Institute of Justice, 1998.

Go Back to The Ripper and The Writers