PMID- 10330204
OWN - NLM
STAT- Publisher
LR  - 20191120
IS  - 1084-8592 (Print)
IS  - 1084-8592 (Linking)
VI  - 1
IP  - 2
DP  - 1996 Jun
TI  - Strategies for Unambiguous Detection of Allelic Heterozygosity via Direct DNA 
      Sequencing of PCR Products: Application to the HLA DRB1 Locus.
PG  - 89-98
AB  - Background: Many genetic loci exhibit substantial heterogeneity: the human 
      leukocyte antigen (HLA) DRB loci include 139 alleles and the cystic fibrosis 
      transmembrane regulator gene more than 500 known mutations. Identification of 
      alleles at these loci is cumbersome with typical molecular diagnostic methods 
      such as hybridization assays or restriction enzyme analysis. Direct DNA 
      sequencing of polymerase chain reaction (PCR) products is a general approach to 
      complex loci that allows detection of any allele within the nucleotide sequence 
      analyzed. However, direct DNA sequence-based unambiguous identification of 
      heterozygous nucleotide positions using PCR templates is a challenging problem. 
      Methods and Results: The ability of direct DNA sequencing methods to accurately 
      identify HLA DRB alleles was assessed. The authors evaluated the performance of 
      modified T7 and Taq DNA polymerases in isothermal and thermal cycle sequencing of 
      PCR products derived from HLA DRB genes in 235 individuals who were potential 
      donors or recipients of bone marrow transplants. The uniformity of peak intensity 
      and ability to identify heterozygous nucleotide positions was similar when either 
      AmpliTaq FS- or Sequenase DNA polymerase-derived electropherograms were prepared. 
      The modified Taq DNA polymerase allowed the use of unpurified, double-stranded 
      PCR templates. Furthermore, this enzyme could be used in less laborious, less 
      costly cycle sequencing assays coupled with automated fluorescent detection 
      methodology. Direct sequencing performed with either enzyme allowed unambiguous 
      identification of DRB1 alleles, resolution of difficult heterozygous 
      combinations, and recognition of new alleles. Conclusions: The direct DNA 
      sequencing methods employed here for HLA allele identification are relatively 
      efficient and semiautomated, and may be reasonably considered as a general 
      approach to other complex molecular diagnostic problems, especially when coupled 
      to simplified sequencing chemistries allowing cycle sequencing.
FAU - Wu, J
AU  - Wu J
AD  - Department of Pathology, University of New Mexico Health Sciences Center, 
      Albuquerque, New Mexico, USA
FAU - Griffith, BB
AU  - Griffith BB
FAU - Bassinger, S
AU  - Bassinger S
FAU - Moehlenkamp, C
AU  - Moehlenkamp C
FAU - Brodie, SG
AU  - Brodie SG
FAU - Wu, Y
AU  - Wu Y
FAU - Gribble, GG
AU  - Gribble GG
FAU - Troup, GM
AU  - Troup GM
FAU - Williams, TM
AU  - Williams TM
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Mol Diagn
JT  - Molecular diagnosis : a journal devoted to the understanding of human disease 
      through the clinical application of molecular biology
JID - 9614965
EDAT- 1996/06/01 00:00
MHDA- 1999/05/18 00:00
CRDT- 1996/06/01 00:00
PHST- 1996/06/01 00:00 [pubmed]
PHST- 1999/05/18 00:00 [medline]
PHST- 1996/06/01 00:00 [entrez]
AID - S1084859296000069 [pii]
AID - 10.1054/MODI00100089 [doi]
PST - ppublish
SO  - Mol Diagn. 1996 Jun;1(2):89-98. doi: 10.1054/MODI00100089.