High Throughput Hla Sequence-Based Typing (Sbt) Utilizing the Abi Prism® 3700 Dna Analyzer

Abstract

Aims and background The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). Methods The SBT method described utilizes an ABI Prism® 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism® 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. Results Data from one 96-well capillary run on the ABI Prism® 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. Discussion The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

HLA-A Gene Polymorphism Defined by High-Resolution Sequence-Based Typing in 161 Northern Chinese Han People

“…We analyzed HLA-A polymorphism in Han and Uygur by sequence specific oligonucleotide probe (SSOP) in the past ( 5 ). With the development of HLA typing technology, the SBT method results in the most possible level of resolution of HLA genotypes 6., 7., 8.. The basic theory of SBT is to get the nucleotide sequence of the most polymorphic regions in the HLA genes and compare the sequence data to those of all possible theoretical allele combinations by software analysis (MatchTool and MT Navigator), which might ensure a highly accurate identification of the actual genotypes of samples ( 11 ).…”

“…4., 5.), sequence-based typing (SBT; ref. 6., 7., 8.) and DNA chip technology 9., 10.. Now, SBT is of more importance as the number of HLA alleles is increasing, and it is a high-resolution and high-throughput typing method compared with SSOP or SSP.…”

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

“…This gave us the opportunity to identify different homo- heterozygous combinations using genomic DNA from previously classified HLA-typed Epstein-Barr virus-transformed B lymphoblastoid (EBV) cell lines obtained from the European Collection of Animal Cell Cultures. The HLA type of all cell lines was verified using sequence-based typing methods [20]. According to conventional display, the HLA-A*0101 sequence was chosen as the consensus .…”

A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping

“…Problems include false negatives or positives in the case of rare or null alleles, and difficulty determining the presence of alleles not specifically assayed for by the panel, which can present misleading amplification or hybridization patterns [20-23]. Novel allele discovery and unambiguous first-pass genotyping requires sequence-based typing (SBT) of individual HLA loci, with current methods using locus- and exon-specific amplification primers, followed by Sanger capillary sequencing [24]. This method, while practical for allele discovery, is underutilized for genotyping due to limitations of Sanger SBT, primarily throughput restriction from multiple time- and cost-intensive exon-specific amplicon preparations each of which must be capillary sequenced [24, 25].…”