In parentage testing, an individual's DNA is used to prove or disprove his or her relatedness to a particular child.
Parentage, or paternity, testing may be sought solely for the sake of curiosity, but is most often used to prove or disprove paternity for legal purposes. Legally motivated reasons to seek parentage testing can include contested child support, custody, or visitation rights, immigration status, adoption, and insurance or inheritance claims.
Historically, paternity testing was based on examination of the blood groups of the child and putative father, and the test could only exclude the possibility of relatedness when blood groups did not match; matching blood groups could not prove parentage, but only the possibility thereof. Modern testing, based on the examination of DNA, can prove with virtually 100% certainty exclusion of paternity or the probability to a certainty of 99.9% that a given person is the parent of a particular child.
Parentage testing is recommended in adoption cases, as identifying both biological parents can help the adoptive parents judge the possibility that the adopted child will develop certain inheritable medical conditions such as cystic fibrosis, or Tay Sachs disease. Further, as the adopted child grows to adulthood, a genetic medical history can prove useful in diagnosis of other conditions such as breast or colon cancer or heart disease. Also, paternity testing ensures that the true biological father relinquishes parental rights and negates the possibility that the adoption will be contested later.
In the infrequent but dramatic cases of babies being switched at birth, parentage testing allows unequivocal identification of parents. In surrogacy cases, DNA testing can confirm the success of the implantation procedure by verifying the identity of the biological parents.
Currently, the vast majority of DNA testing for parentage determination is performed by commercial laboratories that are not associated with a hospital, blood bank, or medical laboratory. The focus of such facilities is often legal rather than medical. Persons seeking testing should be aware that psychological support and genetic counseling may be needed following parentage testing to help them deal with the implications of the results.
Many laboratories advertising paternity testing services lack any accreditation. Individuals seeking paternity testing should choose a laboratory that is accredited by the American Association of Blood Banks (AABB), which performs on site laboratory inspections to ensure that the techniques and equipment being used are acceptable and that the methods followed are consistent with the strict national standards established for paternity/parentage testing.
When blood or other body fluid is collected for DNA analysis, universal precautions should be observed for the prevention of transmission of blood-borne pathogens.
It is important that the parties seeking parentage testing clearly understand their own motivation for testing. Testing that includes "chain of custody" of the samples to be analyzed—notarized proof of identity of all parties being tested and traceable transport of samples from collection to the testing laboratory—is admissible as evidence in the courtroom, but costlier than testing without chain of custody. However, testing on self-collected samples for curiosity purposes, in which chain of custody protocols are not strictly followed, has no legal standing.
The DNA restriction fragment length polymorphisms (RFLPs) and short tandem repeat (STR) loci that commonly serve as identity markers are noncoding regions (i.e., DNA that is not transcribed into RNA and does not code for proteins ). As such, the mutation rate in these sections of DNA tends to be higher than in normal genes. Also, the polymerase chain reaction (PCR) used to amplify the STRs is subject to introduction of mutations. These mutation events can complicate the interpretation of results.
Interpretation of parentage testing results are generally based on the assumption that the alleged father is not related to the actual father, which may not be true. Also, extra care is required in the interpretation of results in the case where the mother's DNA is not available for testing.
Modern parentage (paternity) testing is also known as DNA testing, profiling, or fingerprinting. The dramatic evolution of DNA profiling techniques has been in the field of forensic identification, and since 1985, the technologically advanced DNA-based methodology has essentially completely displaced blood-antigen-based paternity investigation.
Restriction fragment length polymorphisms (RFLPs)
One approach to DNA fingerprinting is based on analysis of slight differences between individuals in the sequence of nucleotides, called sequence polymorphisms, in the chromosomal DNA. A sequence change can cause restriction endonucleases, enzymes used to cut the DNA into pieces small enough to analyze, to make fewer cuts in the DNA, leading to DNA fragments of different sizes called restriction fragment length polymorphisms (RFLPs). These RFLPs are well cataloged, and every person will display a given set of them upon analysis. Clusters of RFLPs tend to be consistent within ethic groups. A greater number of matches between individuals indicates a greater probability of relatedness. Each person has RFLPs inherited from both parents, and thus has a unique RFLP "fingerprint."
In a RFLP DNA analysis, 1-5 ml of blood is drawn from which about 100 ng DNA is extracted and treated with a restriction endonuclease. The DNA fragments are separated by electrophoresis on an agar or polyacrylamide gel, denatured and transferred to nitrocellulose paper, and incubated with pieces of radioactively labeled DNA probes complimentary for the RFLPs. The RFLPs that are present in a sample show up as dark bands on X-ray film exposed to the nitrocellulose sheet.
Short tandem repeats (STRs)
The current state-of-the-art approach to DNA profiling is the investigation of short tandem repeat (STR) loci—short sequences of DNA, normally two to six base pairs that are repeated head to tail numerous times. STRs, also known as microsatellite DNA, are, like RFLPs, well characterized, and each individual carries a distinct set inherited from both parents. STRs are the result of length polymorphisms (inherited differences in the number of these short sequences) as opposed to sequence polymorphisms (inherited differences in the order of bases). The extracted DNA is subjected to amplification in the polymerase chain reaction (PCR), in which fluorescently labeled primer pieces of DNA specific for known STR sequences are incubated with appropriate enzymes and nucleotide building blocks to amplify synthesis of the STR regions of the sample DNA; only the STRs that are present in the sample DNA become amplified. The fluorescently labeled amplified STRs are then separated by gel or capillary electrophoresis, and read by a fluorescence detector. DNA fingerprinting based on STRs has the advantage of being more sensitive than tests based on RFLPs, that is, requiring only 1-5 ng of DNA extracted from a few drops of blood, or from buccal cells collected with a swab from the inside of the cheek. Further, the PCR technique, capillary electrophoresis separation, and fluorescence detection are amenable to automation, leading to faster throughput and less human error.
In 1997, the FBI announced the selection of 13 STR loci to constitute the core of a national DNA profiling database known as CODIS, which has been widely adopted by forensic DNA analysts worldwide. All CODIS STRs are discrete tetrameric repeat sequences that behave according to known principles of population genetics and can be rapidly analyzed with commercially available kits. The CODIS STR set of loci is rapidly becoming the industry standard in paternity testing. A kit that tests the 13 CODIS plus three more STR sites has recently become commercially available.
The laboratories that perform parentage testing are generally commercial facilities engaged in only parentage testing. The person seeking parentage testing contacts such a laboratory to receive instructions. Generally, the appointment for sample collection is scheduled at a local medical laboratory or clinic contracted by the testing laboratory. At the time of scheduling, the names, addresses, and telephone numbers of all persons to be tested, the date of birth or approximate age of the child(ren) to be tested, the preferred day and time for the sample collection, and the name and contact information of any attorney(s) involved is recorded. It is possible to schedule collections for different people in different locations at different times. The samples are all shipped to the testing laboratory to be analyzed simultaneously.
For testing with chain of custody, it is extremely important that everyone being tested is positively identified. For every adult person being tested, the social security number and a picture ID, such as a driver's license, passport, or state identification card, is required and for each child a birth certificate must be provided. Photographs and fingerprints of all persons may be taken at the time of sample collection. Strict chain of custody procedures must be followed, and all information and results are kept strictly confidential and are not released without proper prior authorization.
A small blood sample, usually from a finger prick, is collected from the mother, the child(ren), and alleged father(s). For newborns, the blood sample can be obtained from the umbilical cord at birth, or from a heel prick. Alternatively, cells from the inside of the mouth are collected with a buccal swab. It is also possible to arrange for prenatal testing to be performed on chorionic villi or precultured amniotic fluid cells.
Buccal swabs have become the specimen collection method of choice for DNA testing. The specimen is collected by gently stroking the inner facial cheek with the swab for 30 seconds. It is not necessary to fast before specimen collection, since buccal swab specimens are unaffected by foods, toothpaste, cigarettes, chewing tobacco, lipstick, or bacterial DNA. The collected buccal cells are still usable after years of storage. Buccal samples do not need to be refrigerated nor is immediate shipping to the laboratory required.
To extract DNA from the swab, the head of the swab is transferred to a small plastic tube containing a small amount (0.6 ml) of dilute sodium hydroxide solution (50 mM), and the stick is cut off to allow the tube to be closed (special buccal swabs with ejectable heads have recently become available). The tube is mixed and incubated for five minutes in boiling water, after which the swab head is removed, and a few drops of a buffer (0.06 ml Tris-HCL, pH 8) are added.
For curiosity testing without chain of custody, kits can be obtained for home collection of cheek cells with buccal swabs. The samples are then sent by courier to the testing laboratory.
The fee for testing, with chain of custody, one alleged father and one child, usually with or without the mother, is $400-500, and about $150 is charged for each additional person tested. The level of the fee may also depend on the number of DNA loci or systems probed; generally six to 16 loci are analyzed. Most parentage testing firms require payment of a nonrefundable deposit of $100-150 to initiate the scheduling process; this deposit is applied toward the total fee. Payment may be made by major credit card, certified check, or money order, and is unlikely to be covered by medical insurance. In most cases, the local collection facility additionally charges a specimen collection fee, typically $15-40 per person. The fee for prenatal testing can be substantially higher. Testing without chain of custody can cost $280-400, depending on whether samples are collected at home or by a clinic. Curiosity testing may be performed on samples other than collected blood or buccal cells, such as cigarette butts, chewed gum, bloodstained or semen-stained clothing, used condoms, plucked hair or electric razor debris, or Q-tips containing earwax; additional fees may apply for non-standard samples.
No physical preparation is required. For chain of custody testing, identification documents for every person to be tested must be provided at the time of sample collection.
Amniotic fluid— The watery fluid in the amnion, in which the embryo is suspended; the fluid contains cells of fetal origin.
Buccal— Pertaining to the cheek.
Capillary electrophoresis— A technique for separating biomolecules such as DNA in a fluid-filled thin glass tube on the basis of size and rate of migration in an electric field.
Chorionic villi— Branching outgrowths of the chorion that form the placenta in combination with maternal tissue.
DNA— Deoxyribonucleic acid, a long polymeric biomolecule composed of two self-complementary deoxyribonucleotide strands that adopt a double helical structure and become tightly coiled together with proteins to form chromosomes; DNA is the molecule that stores and transfers the genetic information in virtually all life forms.
Electrophoresis— A technique for separating biomolecules such as DNA in a gel medium on the basis of size and rate of migration in an electric field.
Nucleotide— A biomolecule composed of one of the organic nitrogen-containing bases (adenine, cytosine, guanine, or thymine), a phosphate group, and a pentose sugar that serve as the building blocks of DNA and RNA (in RNA, the thymine base is replaced by uracil).
Polymerase chain reaction (PCR)— A method used in DNA analysis whereby a specific region(s) of the DNA sequence is amplified, allowing rapid DNA analysis.
Polymorphism— The presence of two or more distinct phenotypes in a population due to the expression of different alleles of a given gene.
Restriction endonuclease— Any of a group of enzymes that catalyze the cleavage of DNA molecules at specific sites, used in recombinant DNA technology.
Short tandem repeat— A defined region of DNA, also called microsatellite DNA, containing multiple copies of short sequences of bases repeated a number of times.
Results are returned generally after one to two weeks, and are usually not released until all fees have been collected. Many facilities offer express service with shorter turn-around times, but with correspondingly larger fees, for example, up to about $1,500 for results returned within one working day.
The sample from the child(ren) will give rise to banding patterns on the gel or in the electropherogram reflecting bands inherited from each parent. On the basis of the banding pattern, inclusion or exclusion of parentage is decided.
The commercial parentage testing laboratories typically guarantee over 99.9% exclusion and over 99.0% inclusion of paternity. The actual numbers for the state-of-the-art testing protocols are 100% exclusion and 99.99% inclusion of parentage. For analyses in which the mother's DNA is not available for testing, the rates of inclusion can drop dramatically to 80-99% depending on the number of gene loci examined.
Health care team roles
Initial consultations and scheduling of sample collection appointments is carried out by a representative of the commercial parentage testing facility, often by telephone. A nurse, phlebotomist, or laboratory technician collects the samples and verifies documents at the locally contracted clinic or laboratory, and arranges for transport of the samples to the testing facility. At most accredited parentage testing laboratories, the sample analysis is performed by Ph.D. scientists.
Finis, C. J., Ph.D. "Megaplex STR Analysis from a Single Amplification: Validation of the PowerPlex 16 System." Profiles in DNA (January 2001): 3-6. 〈http://www.promega.com〉.
Lee, H.-S., J. W. Lee, G.-R. Han, and J.-J. Hwang. "Motherless Case in Paternity Testing." Forensic Science International 114, no. 2 (Nov. 13, 2000): 57-65.
Polesky, H. F., M.D. "Impact of Molecular (DNA) Testing on Determination of Parentage." Archives of Pathology and Laboratory Medicine 123, no. 11 (1999): 1060-1062.
Thomson, J. A., V. Pilotti, P. Stevens, k. L. Ayres, and P. G. Debenham. "Validation of Short Tandem Repeat Analysis for the Investigation of Cases of Disputed Paternity." Forensic Science International 100, no. 1-2 (Mar. 15, 1999): 1-16.
DNA Diagnostics Center. 205 Corporate Court, Fairfield, OH 45014. (800) 613-5768. 〈http://www.dnacenter.com/about.html〉.
DNA Testing Centre, Inc. 1201 W. Airport Fwy. STE 255, Euless, TX 76040. 〈http://www.homedna-test.com〉.
GeneTree Inc. 3150 Almaden Expressway #203 San Jose, CA 95118-1253. (408) 723-2670. 〈http://www.genetree.com〉.
The National HLA Fund, Inc. 909 Oradell Avenue, Oradell, NJ 07649-1924, 〈http://www.nationalhlafund.org〉.
Hallick, Richard B. STRs: Short Tandem Repeat Polymorphisms Course web-page, University of Arizona. Copyright 1997–1998; revised Dec. 3, 2000. 〈http://www.u.arizona.edu〉.