TY - JOUR

T1 - Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches

AU - Fugger, L

AU - Morling, N

AU - Ryder, L P

AU - Svejgaard, A

AU - Ødum, Niels

N1 - Keywords: Alleles; Amino Acid Sequence; Base Sequence; DNA Probes, HLA; Genetic Techniques; Genetic Variation; HLA-DP Antigens; Humans; Magnesium Chloride; Molecular Sequence Data; Nucleic Acid Hybridization; Polymerase Chain Reaction; Temperature

PY - 1990

Y1 - 1990

N2 - The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must be carefully titrated for each primer pair in the PCR. The influence of mismatches between the primer and the DNA template were studied and we found that, by using primers differing only from each other at the 3' end, cross-amplification of closely homologous alleles could be avoided. Thus, single base mismatches may be detected in the PCR and typing for HLA-DP gene variants, which differ for only one base, may be performed.

AB - The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must be carefully titrated for each primer pair in the PCR. The influence of mismatches between the primer and the DNA template were studied and we found that, by using primers differing only from each other at the 3' end, cross-amplification of closely homologous alleles could be avoided. Thus, single base mismatches may be detected in the PCR and typing for HLA-DP gene variants, which differ for only one base, may be performed.

M3 - Journal article

C2 - 2191042

VL - 129

SP - 175

EP - 185

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 2

ER -