title: Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.
abstract: Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mitsunaga
authors.forename: Sayoko
authors.initials: S
authors.lastname: Shimizu
authors.forename: Yuko
authors.initials: Y
authors.lastname: Okudaira
authors.forename: Akira
authors.initials: A
authors.lastname: Oka
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tanaka
authors.forename: Minoru
authors.initials: M
authors.lastname: Kimura
authors.forename: Jerzy K
authors.initials: JK
authors.lastname: Kulski
authors.forename: Ituro
authors.initials: I
authors.lastname: Inoue
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Inoko
mesh_terms: D000483:Alleles; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005838:Genotype; D059811:HLA-DRB1 Chains / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011938:Rec A Recombinases / Q000737:chemistry*
chemical_list: D059811:HLA-DRB1 Chains; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes