DNA typing is a laboratory procedure that detects normal variations in a sample of DNA (deoxyribonucleic acid). DNA typing is most often used to establish identity, parentage, family relationship and appropriate matches for transplantation of organs and tissues.
DNA is a molecule that stores genetic information required for the development of the body and the control of cellular processes. Each strand of DNA is made of individual nucleotides that are joined together. Each nucleotide is made up of a phosphate group, a five-carbon sugar (deoxyribose), and an organic base. Adenine, thymine, guanine, and cytosine are the four bases found in DNA. The sequence of bases in DNA determines the genetic code of an individual. Every person excluding identical twins has a different sequence of bases.
The DNA sequence is made up of coding and noncoding regions. A coding region is a section of DNA, called an exon, that contains the instructions for the production of a particular protein. The primary structure of proteins is determined by the sequence of bases in the exons of a gene. Exons are located within the genes but are separated by non-coding regions called introns. Genes make up about 5% of human DNA and the other 95% consists of non-coding regions. The function of the non-coding regions is for the most part unknown.
Normal variations can occur in the DNA sequence of the coding and non-coding regions. Sequence polymorphism and length polymorphism are the two main forms of DNA variation. Sequence polymorphism results from differences in the sequence of bases at a particular locus. A locus is a specific location on a DNA molecule. Length polymorphism results from differences in the length of DNA at a particular locus. Differences in the length of the DNA are due to variations in the number of times that a certain sequence of bases is repeated. The number of times that a certain sequence is repeated at a specific locus will often vary between individuals. A locus that has a repeated unit of nine to ninety-eight bases is called a variable number tandem repeat locus (VNTR) or minisatellite. Loci that have a repeating unit of only two to seven bases are called short tandem repeats (STRs) or microsatellites. For example CAGACAGACAGA is an STR of four bases that is repeated three times.
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.
DNA typing to establish identity, parentage, and family relationship
DNA typing used for identification purposes and the establishment of parentage and family relationships looks for variation within the non-coding regions of the DNA. It relies on the presence of length polymorphisms such as VNTRs and STRs.
DNA typing determines the number of times that a particular sequence is repeated at a particular locus. DNA typing usually analyzes the number of repeats at four or more separate loci. The loci chosen for DNA typing have a high degree of variability and are usually found on different chromosomes.
It is possible that two unrelated individuals will possess the same number of repeats at a particular locus. It is less likely, however, that two unrelated individuals will have the same number of repeats at a number of different loci. In order to determine the possibility that a match in DNA types occurred by chance, the frequency of the determined number of repeats in the individual's ethnic group is calculated. For example, if the frequency of the repeat number in the ethnic group is 1 in 10,000, then there is a 1 in 10,000 chance that the match occurred by chance. By using a number of different loci, one can decrease the chances that the match occurred by chance and increase the chances that the two DNA samples are from the same individual. While it is mathematically impossible to prove that two samples are identical using this procedure, it is possible to prove absolutely that two samples are not identical. This occurs when a different number of repeats is found at a locus.
Mitochondrial DNA typing may be performed if nuclear DNA typing is not successful or when there is insufficient nuclear DNA for typing. It is also sometimes used to help establish maternity. Variations in mitochondrial DNA are typically discovered by sequencing (determining the order of bases).
PARENTAGE AND FAMILY RELATIONSHIP. DNA typing used for establishment of parentage and family relationships uses either RFLP analysis or PCR based STR analysis. DNA typing is the method most often used to establish paternity. It may also be used to establish parentage in cases where neonates have been mistakenly switched in the nursery. DNA typing can be used to resolve immigration cases when family relationships are disputed.
The basis for DNA typing in parentage and family relationship cases is that each child inherits one of each chromosome from their mother and one of each chromosome from their father. The child should, therefore, inherit a set number of repeats at a particular locus from their mother and a set number of repeats at that locus from their father. At some loci they may inherit the same number of repeats from their mother and from their father. During parentage analysis the number of repeats found at different loci is compared between the child and the alleged parent. It is helpful, but not absolutely necessary, to perform DNA typing on the known parent as a comparison. Sometimes the DNA of siblings and other family members will also be typed for comparison. If the alleged parent is the true biological parent, then at each locus analyzed, the child should have the same repeat number as the parent. Since a match can occur by chance it is necessary to calculate the frequency of the repeat number in the alleged parent's ethnic group. Usually when a match is found the chance of parentage is greater than 99%. For example a 1/1000 chance that the match occurred by chance corresponds to a 99.9% chance that the alleged parent is the true biological parent.
If the alleged parent's repeat numbers do not match with the child's repeat numbers then parentage can usually be ruled out. What can make this analysis tricky is that sometimes a small alteration can occur in the DNA of the child that is not present in the parent. This can cause the child to have a different repeat number then their true parent at a specific locus. It is therefore important that more than one loci be analyzed.
Forensic and non-forensic identification
Modern day DNA typing for identification purposes usually uses PCR and STRs rather than RFLP analysis. DNA typing can be used to help identify a victim of a homicide. The DNA type of the victim can be compared to the DNA types of alleged family members to see if there is a match. DNA typing has also been used to identify soldiers killed in battle. The U.S. Department of Defense has a collection of tissue samples from soldiers so that, if necessary, they can be identified through DNA typing.
DNA typing can also help to determine whether a suspect was at a particular crime scene. DNA can be isolated from tissue such as skin, blood, hair and semen left at the scene. The DNA type of the sample at the crime site can be compared to the DNA type of the suspect. If there is a difference in the DNA type at any locus then the DNA obtained is definitely not from the suspect. If the DNA type is identical at all loci then the probability that this occurred by chance must be determined.
DNA typing for transplantation matches looks at normal variation in the coding region of the DNA. DNA typing can be used to identify an appropriate match for a transplant such as a bone marrow transplant. A successful transplant requires a close match of human leukocyte (HLA) antigens between donor and recipient. The HLA antigens are proteins that are located on the surface of most cells. There are six subregions on chromosome number six that each contain at least one HLA locus. The subregions are divided into two classes. HLA-A, HLA-B, and HLA-C comprise Class I and HLA-DR, HLA-DP, and HLA-DQ comprise class II. Each subregion has at least one gene that has a number of different genetic variants (allels). The HLA gene products mediate the recognition and the destruction of foreign cells such as bacteria and viruses . They also mediate the destruction (rejection) of transplanted cells that do not express the same HLA allels as the recepient. Therefore, a transplant from a donor with very different HLA antigens is likely to be rejected by the body's immune system . Conversely, if the donor and recipient have a close HLA match then the transplant has a better chance of being successful.
DNA typing looks for characteristic sequence differences in the genes that produce the HLA antigens. For example, there are more than fifty HLA-A variants. Each HLA-A variant is produced by a different gene. The HLA variants inherited by an individual can be determined by extracting DNA from blood cells and using PCR to amplify the HLA loci. Specific primers or probes are used to identify the variants for each of the six subregions.
Early use of DNA typing for forensic evidence was marred by technical difficulties. By the year 2001, however, DNA typing techniques had improved considerably and had become highly accurate tools for forensic evidence. DNA typing evidence is now widely used in most North American and European courts. The quality of the evidence is still dependent, however on the methodology used, the number of loci examined and the quality of the laboratory where the typing is performed. Contamination of the DNA sample collected from the crime site is also a concern if proper techniques are not followed.
The increasing use of DNA typing has led to the formation of databases that contain DNA typing information. DNA data banks of convicted criminals exist in each of the 50 states and in many other countries. The FBI has also created a national DNA data bank of convicted criminals called CODIS (combined DNA index system). These DNA data banks require convicted felons to donate blood samples for DNA typing prior to parole. The type of felonies for which DNA typing is required varies from state to state. It is anticipated that most laws will be amended to require DNA typing of all convicted felons. It is also possible that laws may be enacted to force all people arrested for a crime to donate a DNA sample to the database.
Many countries obtain DNA samples from ordinary citizens when trying to eliminate suspects of a particular
Amniocentesis —Prenatal testing performed at 16-20 weeks of pregnancy which involves inserting a needle through the abdomen of a pregnant mother and obtaining a small sample of fluid from the amniotic sack. Can be used to obtain a sample of the baby's cells for DNA typing.
Antibody —Protein produced by the body in response to the presence of a foreign antigen.
Antigen —A substance that induces an immune response.
Chorionic villus sampling (CVS) —Prenatal testing performed at 10-12 weeks of pregnancy which involves inserting a catheter through the vagina of a pregnant mother or inserting a needle through the abdomen of the mother and obtaining a sample of placenta. Can be used to obtain a sample of cells for DNA typing.
Chromosome —A microscopic structure, made of a complex of proteins and DNA, that is found within each cell of the body.
DNA (deoxyribonucleic acid) —The hereditary material that influences the development and functioning of the body.
Eugenics —A discredited movement which attempts to improve the human race by preventing the creation of offspring in individuals with undesirable traits and promoting the creation of offspring in those with desirable traits.
Forensic —Related to or used in the courts of law.
Gene —A functional segment of DNA that contains the instructions for the production of a particular protein. Each gene is found on a specific location on a chromosome.
HLA typing —The determination of the type of human leukocyte antigens possessed by an individual.
Locus —Specific physical location on a DNA molecule.
Nucleus —A membrane bound spherical structure that contains the chromosomes and is found in most cells.
Polymorphism —Genetic variation.
STR (short tandem repeats) —A locus of DNA that has a repeating unit of two to seven bases.
VNTR (variable number tandem repeats) —A locus of DNA that has a repeating unit of 9 to 98 bases.
crime. For example DNA samples may be collected on hundreds of men in a rural area where a sexual assault has occurred. Although citizens are not required by law to donate their DNA, it is likely that they experience a great deal of social coercion. The United States does not currently allow DNA typing of those who are not convicted of a crime. There are concerns, however, that the databases could be expanded to include all citizens and be used as a method of identification similar to a social security number.
Commercial databanks exist which store DNA samples or DNA typing results of children. If the child is later kidnapped then DNA samples obtained from such items as gum and licked stamps can be used to try and locate and identify the child. Some people would argue that these databanks are unnecessary since a child's DNA type can be deduced from that of other family members should it become necessary. This stored information could also be used later as evidence against the child. If the commercial data bank becomes bankrupt or changes ownership then there may be concerns about the control, availability and ownership of the DNA. In addition, issues of non-paternity can be discovered during this process.
The existence of DNA databanks raise concerns about privacy and discrimination. There is a concern that DNA typing may yield other unintended genetic information about the individual and his or her family. There has been some discussion about donating DNA samples that have been collected for forensic DNA typing to researchers. This research could yield very specific genetic information on the individual. There is a concern that the genetic information obtained could be used for discriminatory purposes. For example, the information could result in the discrimination of citizens by employers and health insurance companies.
DNA typing results often become evidence in a court of law. It is, therefore, important that health care professionals involved in collecting samples for DNA typing follow strict protocols that prevent contamination and insure that samples are obtained from the correct person. It is also important that a chain-of-evidence is established. A chain-of-evidence documents who has handled the evidence and when it was transferred to another person. A chain-of-evidence can help to demonstrate that evidence was not altered prior to its introduction in court. Often, laboratories that perform DNA typing for parentage have kits designed to insure that proper protocols are followed. They may also have designated blood collection centers that will obtain the samples required for DNA typing. Many hospitals have specific protocols, kits, and chain-of-evidence procedures in place to insure that samples for DNA typing are collected and handled properly. In some cases special training programs are offered to health care professionals who are involved in collecting samples for DNA typing used as forensic evidence.
It is important that health care professionals try to help prepare their patients for paternity test results since the results can sometimes be unexpected and can often be quite devastating. It can be helpful to explore the motivation behind the testing and discuss the implications of possible test results. The limitations of the testing should also be adequately discussed. The quality of the laboratory chosen for paternity testing should also be evaluated.
Some health care professionals may be called upon to offer paternity testing prenatally. The patient needs to be informed about the risks of losing a normal pregnancy if prenatal testing methods such as amniocentesis and chorionic villus sampling are performed. It is also typically more expensive to perform paternity testing prenatally. Many patients when provided with complete information about prenatal paternity testing choose to have the tests performed after the child is born.
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Lisa Maria Andres, MS, CGC