PCR-sequence-specific oligonucleotide probing

Related Terms

Allele, forensic medicine, HLA, HLA class I antigens, HLA class II antigens, HLA locus, human leukocyte antigens, human lymphocyte antigens, PCR, PCR-based HLA typing, PCR-sequence-specific oligonucleotide probing, PCR-sequence-specific primer, PCR-SSO probing, PCR-SSP, polymerase chain reaction, polymorphism, primer, serotyping, SSO, SSP, transplant.

Background

Human lymphocyte antigens (HLA), or human leukocyte antigens, are proteins found on the surface of almost all the cells in the body. Proteins are a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur, and they are composed of one or more chains of amino acids. Amino acids are the building blocks of proteins. HLA are present in especially high concentration on white blood cells, which play an important role in the body's immune response to foreign substances. Each individual has a unique set of HLA genes, as both parent pass down one of these genes to their offspring. Identical twins, however, can have the same set of HLA genes.
These HLA genes code for the HLA, which play a vital role in organ transplant success. A transplant is the transfer of an organ such as kidney or liver from one person (the donor) to another (the recipient) when the recipient's own organ is severely damaged. The donor's and recipient's HLA should be as similar as possible to ensure transplant success. Therefore, transplantation of an organ from an identical twin will be more successful than between siblings. Transplantation from an unrelated donor may be less successful than from a sibling.
The genetic information in humans is passed down from the parents to their offspring through 23 pairs of chromosomes. Each chromosome consists of a long deoxyribonucleic acid (DNA) molecule and associated proteins that carry part of the hereditary information of an organism. Each DNA molecule itself consists of large numbers of nucleotides. Nucleotides in DNA are composed of a nitrogen-containing base, a 5-carbon sugar (deoxyribose), and phosphate groups. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information. A region of DNA that controls a specific hereditary characteristic, usually corresponding to a single protein, is called a gene. A gene may exist in two or more forms or versions, and these forms are called alleles.
An antigen can be defined in two contexts: A protein marker on the surface of cells that identifies the cell as "self" or "non-self," in which case they stimulate the production of antibodies by B lymphocytes (a type of white blood cell), which can neutralize or destroy the antigen-bearing cell. Antigens may also be foreign substances such as bacteria, toxins, or foreign blood cells, which trigger the body to produce antibodies. Antibodies are proteins manufactured by the body that bind to an antigen to neutralize, inhibit, or destroy it.
HLA loci: "Loci" is the plural of "locus," which is the position of a gene on a chromosome. A chromosome is a single DNA molecule that contains many genes. Humans possess 23 pairs of chromosomes, and the HLA locus is located on the short arm of chromosome 6, which codes for the HLA molecules. The HLA molecules bind and present antigens in the form of peptides to T lymphocytes. Peptides are short chains of amino acids. T lymphocytes, which are a type of white blood cell, then destroy the antigen, which may be a bacterium, a virus, or an infected or malfunctioning cell.
HLA is divided into two groups: Class I and II. HLA class I antigens (HLA-A, HLA-B, and HLA-C) are found on most cells that possess a nucleus and present antigens (usually from a virus or parasite) from inside the cell to the T lymphocytes. They are also found freely in plasma (the liquid part of blood) and on the surface of platelets. Platelets are cells found in blood that play a major role in blood clotting.
HLA class II antigens (HLA-DR, HLA-DP, and HLA-DQ) are glycoproteins present on cells such as B cells and macrophages, and they also present antigens outside the cell to T lymphocytes. B cells, macrophages, and T lymphocytes are types of white blood cells, and a glycoprotein is any of a group of conjugated proteins having a carbohydrate as the non-protein component. A conjugated protein consists of amino acids and carbohydrates, which are chemical compounds in food that contain only carbon, hydrogen, and oxygen, and include all sugars, starches, and plant fiber (cellulose). A third type, called HLA class III, has functions related to class I and II components. HLA class III codes for proteins related to the complement system, which is involved in clearing pathogens from the body.
HLA typing: HLA typing is a test done in order to determine if a patient has antibodies against a potential organ donor's HLA. HLA typing is also used to establish paternity (to determine if a particular person is the father of a child) and to study disease association; it is also used in forensic medicine. Forensic medicine is the relation and application of scientific techniques to resolve legal matters. For example, DNA extracted from evidence, such as hair or blood from a crime scene, can be matched with DNA from suspects to identify the culprit.
The test was originally performed by serotyping, which is the use of specific antibodies to identify specific antigens. This test required lymphocyte preparations, which are a type of white blood cell that contain the HLA antigen, and specific antibodies to identify these antigens. However, due to the poor availability of specific antibodies and differences in the accuracy of test results among various laboratories, other methods of HLA typing were developed. The serotype-based method of HLA typing has been replaced by DNA-based analysis. DNA-based analysis identifies the sequence of genes that code for the HLA antigens. With the advent of polymerase chain reaction (PCR), a new tool was available for HLA typing.
HLA typing by PCR-based techniques: PCR-based HLA typing uses the polymerase chain reaction (PCR) for HLA typing. PCR is a method that uses enzymes, which are proteins that catalyze biochemical reactions, for the amplification of the two strands of deoxyribonucleic acid (DNA) of a particular gene sequence. Amplification of the DNA allows the analysis of extremely small amounts of a sample containing the DNA. DNA is a long thread-like molecule that carries genetic (hereditary) information. This method has several advantages over the serological techniques in HLA typing. PCR-based techniques are more accurate and precise than serology. They can be done on small samples, such as fingernail scrapings, or dried blood; serology requires 20 to 30 milliliters of fresh blood. Also, PCR-based typing can detect small differences in an allele, which can make a person prone or resistant to developing a disease.

Methods

General: Human lymphocyte antigen (HLA) typing is a test done in order to determine if a patient has antibodies against a potential organ donor's HLA. HLA typing is also used to establish paternity (to determine if a particular person is the father of a child), and to study disease association; it is also used in forensic medicine. Forensic medicine is the relation and application of scientific techniques to resolve legal matters. For example, deoxyribonucleic acid (DNA) extracted from evidence, such as hair or blood from a crime scene, can be matched with DNA from suspects to identify the culprit.
The two main polymerase chain reaction (PCR)-based methods used for human HLA typing are PCR-sequence-specific oligonucleotide (SSO) probing and PCR-sequence-specific primer (SSP).
PCR-sequence-specific oligonucleotide (SSO) probing: An oligonucleotide is a short segment of riboneucleid acid (RNA) or DNA, usually with fewer than twenty bases. In this method, the DNA from the sample is amplified by PCR. Amplification refers to the production of millions of copies of a specific DNA sequence. DNA is a long thread-like molecule made up of large numbers of nucleotides. Nucleotides in DNA are composed of a nitrogen-containing base, a 5-carbon sugar (deoxyribose), and phosphate groups. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information. RNA is a polymeric constituent of all living cells and many viruses, consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases adenine, guanine, cytosine, and uracil bonded to the ribose.
PCR is a method that uses enzymes, which are proteins that catalyze biochemical reactions, for the repeated copying of the two strands of DNA of a particular gene sequence. The amplified DNA is immobilized on a nylon membrane. Then, labeled probes, which are single-stranded pieces of DNA (or RNA) complementary to part of the DNA fragment of interest (in a sample), are hybridized (joined) to the amplified DNA. The probes may be labeled with horseradish peroxidase (an enzyme) or with biotin (a vitamin). The labels help in the detection of the probes. The bound probe is then detected with an enhanced chemiluminescence system, which makes use of the production of light from a chemical reaction, with minimal heat production.
PCR-sequence-specific primer (SSP): This technique makes use of primers that are complementary to HLA polymorphisms, which are two (or more) forms of a gene, with each form being too common to be due merely to new mutation (a change in a gene). Primers are strands of nucleic acid that serve as a starting point of DNA replication. DNA replication is the process by which two double-stranded DNA molecules are produced from a single double-stranded DNA molecule. If a primer is complementary to the sample DNA, a PCR product is produced that can be identified after electrophoresis. Electrophoresis is the movement of charged particles through a medium in which they are scattered under the influence of an electric field. It is used in the analysis and separation of proteins. If the primer is not complementary to the sample DNA, no PCR product is produced.

Research

Research is being carried out to identify an association between human lymphocyte antigen (HLA) and diseases, such as cancer and hepatitis. HLA proteins are found on the surface of almost all cells in the body. Proteins are a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur, and are composed of one or more chains of amino acids. Amino acids are the building blocks of proteins. HLA are present in especially high concentration on white blood cells, which play an important role in the body's immune response to foreign substances. Knowledge of the association between HLA and disease may assist in the early diagnosis and treatment of a particular disease.
For example, an association has been found between the HLA-A2 allele and prostate cancer in Swedish patients. In HLA-A2, A represents the locus and 2 is the actual antigen at the locus. A locus is the position of a gene on a chromosome. A gene may exist in two or more forms, and these forms are called alleles. A recent study has focused on the association between HLA class II antigens and how prone a fetus is to hepatitis B infection in the uterus, when the mother herself carries the infection. HLA class II antigens (HLA-DR, HLA-DP, and HLA-DQ) are glycoproteins present on cells such as B cells and macrophages, and they also present antigens outside the cell to T lymphocytes. B cells, macrophages, and T lymphocytes are types of white blood cells, and glycoproteins are any of a group of conjugated proteins having a carbohydrate as the nonprotein component. A conjugated protein consists of amino acids and carbohydrates, which are chemical compounds in food that contain only carbon, hydrogen, and oxygen, and include all sugars, starches, and plant fiber (cellulose). Hepatitis B is a viral infection of the liver that may cause permanent liver damage if it becomes chronic.

Implications

Organ transplantation: Organ transplantation is the transfer of an organ such as a kidney or a liver from one person (the donor) to another (the recipient). It is done when the recipient's organ is severely damaged. Prior to transplantation, the donor and the recipient should be HLA-matched so that there is less of a chance that the donor's organ will be rejected by the recipient's immune system. Human leukocyte antigen (HLA) matching is the comparison of the HLA of the recipient with the HLA of the donor. For a successful transplant, the donor's and the recipient's HLA should be as similar as possible. Differences in the HLA will make the recipient's body recognize the donor's organ as 'foreign' and reject it. HLA matching is, at present, done by PCR-based HLA typing, a procedure which uses the polymerase chain reaction (PCR) for HLA typing. PCR is a method that uses enzymes, which are proteins that catalyze biochemical reactions, for the amplification of the two strands of deoxyribonucleic acid (DNA) of a particular gene sequence. Amplification of the DNA allows for the analysis of extremely small amounts of a sample containing the DNA. DNA is a long thread-like molecule that carries genetic (hereditary) information.
Studying HLA polymorphisms, which are the existence of two (or more) forms of a gene with each form being too common to be due merely to a new mutation (a change in a gene, in other words, a change in the sequence of base pairs in the DNA that makes up a gene), has provided an understanding of why a foreign tissue gets rejected. Trials have shown that PCR-based typing can increase the one-year transplant rate by more than 20% compared to serological techniques. Serological techniques refer to the use of specific antibodies to identify specific antigens. An antigen can be defined in two contexts: A protein marker on the surface of cells that identifies the cell as "self" or "non-self", in which case it stimulate the production of antibodies by B lymphocytes (a type of white blood cell), which can neutralize or destroy the antigen-bearing cell. Antigens may also be foreign substances such as bacteria, toxic substances, and foreign blood cells that trigger the body to produce antibodies. Antibodies are proteins manufactured by the body and that bind to an antigen to neutralize, inhibit, or destroy it.
Disease association: The class II antigens have been associated with more than 40 diseases. HLA class II antigens (HLA-DR, HLA-DP, and HLA-DQ) are glycoproteins present on cells such as B cells and macrophages, and they also present antigens outside the cell to T lymphocytes. B cells, macrophages, and T lymphocytes are types of white blood cells, and glycoproteins are any of a group of conjugated proteins having a carbohydrate as the nonprotein component. A conjugated protein consists of amino acids and carbohydrates, which are chemical compounds in food that contain only carbon, hydrogen, and oxygen, and include all sugars, starches, and plant fiber (cellulose). The presence of certain HLA alleles is associated with particular diseases. A gene may exist in two or more forms or versions; these forms are called alleles. For example, an association has been found between psoriasis (a skin disorder) and HLA-Cw6; narcolepsy (a sleep disorder) and HLA-DR2 and HLA-DQB1 0602; and multiple sclerosis (a nerve disorder) and HLA-DR2. The letters which follow HLA (for example, DR) indicate the locus, and the numbers refer to the actual antigen at the locus. A locus is the position of a gene on a chromosome. The HLA system, along with other genes, is believed to influence the way an individual responds to environmental factors that can cause disease.
Forensic science: HLA antigens are highly polymorphic, which means that the gene coding for them exists in many forms, and they are, therefore, useful in forensic medicine, the relation and application of scientific techniques to resolve legal matters. This is so because polymorphisms allow a gene to be present in several forms, making each individual possess a unique set of genes. Paternity testing (a test to determine if a particular person is the father of a child) and typing of samples obtained from a crime scene are the two main applications of HLA typing in this field. Quantities as small as few micrograms of a sample, such as hair, fingernail scrapings, semen, or fluid from the vagina, are sufficient for PCR-based typing, unlike the older serological techniques, which required 20-30 milliliters of fresh blood for the test.

Limitations

Polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) typing is a procedure that uses the PCR. PCR is a method that uses enzymes, which are proteins that catalyze biochemical reactions, for the amplification of the two strands of deoxyribonucleic acid (DNA) of a particular gene sequence. Amplification of the DNA allows the analysis of extremely small amounts of a sample containing the DNA. DNA is a long thread-like molecule that carries genetic (hereditary) information. PCR-based typing is highly specific, that is, specific alleles are identified, and there is no cross-reactivity. A gene may exist in two or more forms called alleles. Cross-reactivity is the identification of an allele that is similar to the actual allele of interest. This characteristic is the major advantage of PCR-based typing; however, it is also a disadvantage because new alleles (that are not in the HLA sequence databank) will not be identified. The primers used for the HLA-typing procedure are based on an HLA sequence databank, which contains alleles that were available at the time the databank was designed. Primers are strands of nucleic acid that serve as a starting point of DNA replication. DNA replication is the process by which two double-stranded DNA molecules are produced from a single double-stranded DNA molecule. It is important to regularly update the databank to identify new alleles.

Future research

Human leukocyte antigen (HLA) typing is finding application in the diagnosis of molar pregnancy. A molar pregnancy is a pregnancy marked by a cancer in the uterus, whereby part or all of the chorionic villi (a component of the embryo, which is the early stage of the fetus) are converted into a mass of clear vesicles. HLA typing can also differentiate between different types of molar pregnancy.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography

Alaez C, Lin L, Flores-A H, Vazquez M, et al. Association of narcolepsy-cataplexy with HLA-DRB1 and DQB1 in Mexican patients: a relationship between HLA and gender is suggested. BMC Med Genet. 2008 Aug 15;9:79.
Bugawan TL, Klitz W, Alejandrino M, et al. The association of specific HLA class I and II alleles with type 1 diabetes among Filipinos. Tissue Antigens. 2002 Jun;59(6):452-69.
Claas FH. HLA antibody testing: a tool to facilitate not to prevent organ transplantation. Int J Immunogenet. 2008 Sep 5.
Gyllensten U, Allen M. PCR-based HLA class II typing. PCR Methods Appl. 1991 Nov;1(2):91-8.
Hao GQ, Xiao LL. The application of molecular biology techniques in HLA-typing. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2002 Jun;10(3):268-72.
Li D, Zhang LL, Ju XL, et al. Study of single cell PCR for HLA typing. Zhonghua Xue Ye Xue Za Zhi. 2007 May;28(5):308-11
Natural Standard: The Authority on Integrative Medicine. .
Petersdorf EW, Hansen JA, Martin PJ, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med. 2001 Dec 20;345(25):1794-800
Shankarkumar U, Pawar A, Ghosh K. Implications of HLA sequence-based typing in transplantation. J Postgrad Med. 2008 Jan-Mar;54(1):41-4.
Tiercy JM. Molecular basis of HLA polymorphism: implications in clinical transplantation. Transpl Immunol. 2002 May;9(2-4):173-80.