PMID- 23474534
OWN - NLM
STAT- MEDLINE
DCOM- 20130919
LR  - 20211021
IS  - 1432-1211 (Electronic)
IS  - 0093-7711 (Linking)
VI  - 65
IP  - 6
DP  - 2013 Jun
TI  - Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using 
      RecA and a restriction enzyme for enhanced amplification specificity.
PG  - 405-15
LID - 10.1007/s00251-013-0690-0 [doi]
AB  - 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.
FAU - Mitsunaga, Shigeki
AU  - Mitsunaga S
AD  - Department of Molecular Life Science, Division of Basic Medical Science and 
      Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, 
      Japan.
FAU - Shimizu, Sayoko
AU  - Shimizu S
FAU - Okudaira, Yuko
AU  - Okudaira Y
FAU - Oka, Akira
AU  - Oka A
FAU - Tanaka, Masafumi
AU  - Tanaka M
FAU - Kimura, Minoru
AU  - Kimura M
FAU - Kulski, Jerzy K
AU  - Kulski JK
FAU - Inoue, Ituro
AU  - Inoue I
FAU - Inoko, Hidetoshi
AU  - Inoko H
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130309
PL  - United States
TA  - Immunogenetics
JT  - Immunogenetics
JID - 0420404
RN  - 0 (HLA-DRB1 Chains)
RN  - EC 2.7.7.- (Rec A Recombinases)
RN  - EC 2.7.7.7 (DNA-Directed DNA Polymerase)
RN  - EC 3.1.21.- (DNA Restriction Enzymes)
SB  - IM
MH  - Alleles
MH  - DNA Restriction Enzymes/*chemistry
MH  - DNA-Directed DNA Polymerase/chemistry
MH  - Genotype
MH  - HLA-DRB1 Chains/chemistry/*genetics
MH  - Humans
MH  - Molecular Sequence Data
MH  - Nucleic Acid Amplification Techniques/*methods
MH  - Rec A Recombinases/*chemistry
EDAT- 2013/03/12 06:00
MHDA- 2013/09/21 06:00
CRDT- 2013/03/12 06:00
PHST- 2012/12/25 00:00 [received]
PHST- 2013/02/15 00:00 [accepted]
PHST- 2013/03/12 06:00 [entrez]
PHST- 2013/03/12 06:00 [pubmed]
PHST- 2013/09/21 06:00 [medline]
AID - 10.1007/s00251-013-0690-0 [doi]
PST - ppublish
SO  - Immunogenetics. 2013 Jun;65(6):405-15. doi: 10.1007/s00251-013-0690-0. Epub 2013 
      Mar 9.