DNA Typing Health Article

Nuclear and mitochondrial DNA

Two strands of single stranded DNA wind together to form double stranded DNA in the form of a double helix. The DNA strands are held together by hydrogen bonds that form between the bases. Adenine joins with thymine and guanine joins with cytosine. Two sequences are said to be complementary if they have a sequence that allows them to join together to form double stranded DNA.

Most DNA is packaged with proteins to form microscopic structures called chromosomes. Chromosomes are found in the nucleus of each cell of the body and can be visualized under the microscope. Each cell of the body, except for the egg and the sperm cells, normally contains 22 pairs of chromosomes and two sex chromosomes (46 chromosomes in total). The egg and sperm cells each contain 23 chromosomes.

DNA is also found in the mitochondria. The mitochondria are energy producing organelles found in most cells. There are many mitochondria found in each cell. Each mitochondria contains one copy of circular DNA. Since there are many mitochondria in each cell, a lot of mitochondrial DNA may be present in only a small sample of cells. Mitochondrial DNA is found in the egg cells but not the sperm cells. Mitochondrial DNA is, therefore, only passed down from a mother to her offspring.

Methods of DNA typing for identity, parentage, and family relationships

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSIS. RFLP analysis was the first technique used for forensic DNA typing. During RFLP analysis, DNA that has been isolated from a sample is cut into short sections by an enzyme called a restriction endonuclease. An enzyme is selected that will make a cut in the DNA at a specific sequence of bases on either side of a VNTR locus. This results in different sized fragments of DNA. Different people will have fragments of DNA of different lengths due to differences in the number of repeating units at a particular DNA locus. After the DNA is cut into pieces, the fragments are separated according to size by a process known as gel electrophoresis. DNA fragments are negatively charged at an alkaline pH owing to the phosphate groups. The smaller the fragment, the faster it will migrate to the positive electrode (anode). After separating the DNA fragments, the gel is soaked in a solution of sodium hydroxide and sodium chloride which separates the double stranded DNA into single stranded DNA, a process called denaturation. The single stranded DNA is transferred from the gel to a nylon membrane. A piece of DNA called a probe is added to the DNA that is affixed to the membrane. The probe will attach to a section of DNA on the membrane that has a complementary base sequence. The probe chosen corresponds to a specific locus that has a variable number of repeats containing the bases complementary to the probe. Either a florescent or radioactive material is bound to the probe so that it can be visualized. Either one or two bands will be visualized for each probe used. If the person has inherited the same length DNA fragment at a locus from both parents then only one band will be seen. If two different sized DNA fragments are inherited then two bands will be visualized. The DNA is analyzed using a panel of probes each specific for a different locus. A DNA type is made up of a pattern of different sized bands at different loci. The frequencies of the VNTRs that are inherited at each locus determines the probability of a DNA match.

POLYMERASE CHAIN REACTION (PCR). PCR based STR analysis is a more modern approach to DNA typing. The first step of the process is to isolate DNA from a sample of tissue such as blood or semen. The isolated DNA, called the template, is mixed with a heat-stable form of DNA polymerase (e.g., Taq polymerase), deoxynucleotide triphosphates, and DNA primers. These reagents are suspended in a buffer and together are called the master mix. The template and master mix are added to a tube and the tube is placed into an incubator called a thermal cycler. The purpose of the thermal cycler is to control the temperature of each of the three phases of DNA amplification.

The first step in PCR is denaturation. This is accomplished by heating the mixture to 94°C, which separates the strands. After denaturation, the temperature of the thermal cycle is automatically lowered to 64°C. The primers are short sequences of DNA that can bind to one of the DNA strands on either side of the target sequence to be amplified. Four primers are required for each section of DNA that is being analyzed. The primers are selected so that they flank an STR region. The binding of the primers to complementary base sequences on the target DNA is called annealing, and requires a lower temperature. After the annealing step, the temperature is increased to 72°C, which is the optimal temperature for DNA polymerase. Once the primers have annealed, the DNA polymerase binds to single stranded DNA between the primers and fills in the sequence with the deoxynucleotide triphosphates. This results in synthesis of a new piece of double stranded DNA consisting of the STR locus and the primer regions. This process is called primer extension. After extension, the new double strands of target DNA are denatured by heating the mixture to 94°C again. After a suitable incubation, the temperature is lowered to 64°C to facilitate annealing of primers, and then is adjusted to 72°C for extension of new complementary strands. Each cycle increases the number of DNA pieces containing the STR locus. Each time this process is repeated the amount of target DNA doubles. After 20 cycles (about two hours) there will be over one million identical copies of the STR locus. DNA typing for identification, parentage and familial studies uses probes that encompass various STR loci. If a person has inherited the same number of repeats from his or her mother and father, then only one band will appear for that locus. If the person has inherited a different number of repeats from his or her mother and father, then two bands will be seen for that locus.