Biomaterial Database

Optimizing PCR conditions for HLA class II SSO typing. 1991

A J Ivinson

North West Regional Tissue Typing Laboratory, St Mary's Hospital, Manchester, U.K.

The polymerase chain reaction (PCR) has made the technique of sequence-specific oligonucleotide (SSO) typing fast, accurate and very sensitive. These combined techniques are an ideal tool for analysing the complex patterns of polymorphism seen throughout the HLA complex. The success of the technique relies heavily on accurate and specific amplification of the DNA under study. This paper considers the principles behind the PCR amplification technique and discusses the factors which lead to optimal amplification. Primer design is discussed and a variety of sources of target DNA considered. Precautions designed to prevent contamination are discussed. Reaction components are considered both in isolation and as part of the complete reaction. Finally, a complete PCR protocol is suggested. The paper is illustrated with examples of HLA class II amplification.

UI	MeSH Term	Description	Entries
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004259	DNA-Directed DNA Polymerase	DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.	DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D004340	Drug Contamination	The presence of organisms, or any foreign material that makes a drug preparation impure.	Drug Adulteration,Drug Contamination, Chemical,Drug Contamination, Microbial,Drug Contamination, Physical,Drug Impurity,Adulteration, Drug,Chemical Drug Contamination,Chemical Drug Contaminations,Contamination, Chemical Drug,Contamination, Drug,Contamination, Microbial Drug,Contamination, Physical Drug,Contaminations, Chemical Drug,Contaminations, Microbial Drug,Contaminations, Physical Drug,Drug Adulterations,Drug Contaminations,Drug Contaminations, Chemical,Drug Contaminations, Microbial,Drug Contaminations, Physical,Drug Impurities,Impurity, Drug,Microbial Drug Contamination,Microbial Drug Contaminations,Physical Drug Contamination,Physical Drug Contaminations
D006650	Histocompatibility Testing	Identification of the major histocompatibility antigens of transplant DONORS and potential recipients, usually by serological tests. Donor and recipient pairs should be of identical ABO blood group, and in addition should be matched as closely as possible for HISTOCOMPATIBILITY ANTIGENS in order to minimize the likelihood of allograft rejection. (King, Dictionary of Genetics, 4th ed)	Crossmatching, Tissue,HLA Typing,Tissue Typing,Crossmatchings, Tissue,HLA Typings,Histocompatibility Testings,Testing, Histocompatibility,Testings, Histocompatibility,Tissue Crossmatching,Tissue Crossmatchings,Tissue Typings,Typing, HLA,Typing, Tissue,Typings, HLA,Typings, Tissue
D006681	HLA-D Antigens	Human immune-response or Class II antigens found mainly, but not exclusively, on B-lymphocytes and produced from genes of the HLA-D locus. They are extremely polymorphic families of glycopeptides, each consisting of two chains, alpha and beta. This group of antigens includes the -DR, -DQ and -DP designations, of which HLA-DR is most studied; some of these glycoproteins are associated with certain diseases, possibly of immune etiology.	Antigens, HLA-D,Class II Human Antigens,HLA-Dw Antigens,Human Class II Antigens,Ia-Like Antigens, Human,Immune Response-Associated Antigens, Human,Immune-Associated Antigens, Human,Immune-Response Antigens, Human,HLA-D,HLA-Dw,Immune Response Associated Antigens, Human,Antigens, HLA D,Antigens, HLA-Dw,Antigens, Human Ia-Like,Antigens, Human Immune-Associated,Antigens, Human Immune-Response,HLA D Antigens,HLA Dw Antigens,Human Ia-Like Antigens,Human Immune-Associated Antigens,Human Immune-Response Antigens,Ia Like Antigens, Human,Immune Associated Antigens, Human,Immune Response Antigens, Human
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain