Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Class I sequence based typing of HLA-A, -B, and -C alleles by direct DNA sequencing

a technology of hla-a, -b, and -c alleles, applied in the field of direct dna sequencing of hlaa,b, andc alleles, can solve the problems of high level of allelic diversity, mhc polymorphism exerting immunological burden on the transplanted host, and a formidable task of hla class i typing at the nucleic acid level

Inactive Publication Date: 2007-06-07
HILDEBRAND WILLIAM H +3
View PDF9 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, MHC polymorphism exerts an immunological burden on the host transplanted with allogeneic tissues.
This high level of allelic diversity complicates the typing of the HLA class I genes.
Taken together, these two complications make HLA class I typing at the nucleic acid level a formidable task.
This approach is not ideal, however, since the primers hybridize with portions of the coding strand, and thus may mask significant allelic variations.
Prior to inclusion of polymerase chain reaction (PCR) into the cloning and sequencing of HLA class I alleles the accumulation of class I sequences was a cumbersome act.
This advance in class II typing was essential because alloantisera, traditionally used for class I typing, proved especially inadequate for typing class II antigens.
The first difficulty is that sequence based typing must frequently resolve two nucleotides at one rung of the sequencing ladder; heterozygosity is the norm, and two alleles at one locus may differ by as many as 85 nucleotides.
The second difficulty is the occurrence of band compressions in the sequencing ladder due to the high G / C content of class I molecules.
A third difficulty encountered by all who type class I HLA molecules is their polymorphic nature; once the class I DNA sequence is obtained, it is often difficult to assign a class I type to the data generated.
However, clinical HLA class I typing laboratories (which now use antibodies for typing) cannot accurately discriminate among the many different class I genes found in the population.
The reason others have been reluctant to adopt a sequence based typing approach is because the technology is complex and developing.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Class I sequence based typing of HLA-A, -B, and -C alleles by direct DNA sequencing
  • Class I sequence based typing of HLA-A, -B, and -C alleles by direct DNA sequencing
  • Class I sequence based typing of HLA-A, -B, and -C alleles by direct DNA sequencing

Examples

Experimental program
Comparison scheme
Effect test

example i

[0036] Once a sample is obtained, the first step is to treat the tissue sample so as to obtain nucleic acids for amplification. The method herein described can be performed on whole blood, tumor cells, sperm, hair follicles, or any other nucleated tissue sample.

[0037] A. Locus Specific Amplification of HLA-A, -B, -C Alleles and of Exons 2 and 3

[0038] Genomic DNA was extracted from 200 μL of whole blood using the Qiagen DNA extraction kit otherwise known as a “QIAamp blood kit” according to the supplied protocol. Exons 2 and 3 of HLA class I-A, -B, and -C loci were PCR amplified from 500 ng of genomic DNA using 20 picomoles of HLA class I locus specific primers located within introns 1 and 3 (Table 1).

[0039] The primers listed in Table 1 correspond to the bases which are the same across the introns and are indicated as a single base (A, C, G, T), while bases which are variable across the introns are indicated by a code for alternative bases. In general, it will be advantageous to ...

example 2

[0076] We first obtained cells for category two typing. The cells we received required HLA-A, -B, and -C nucleotide sequence analysis. After thawing and resuspending the cells in RPMI 1640 supplemented with 15% fetal calf serum, 10 U / ml penicillin, 10 mg / ml streptomycin, and 2 mM L-glutamine, the cells were counted and cell viability was determined using trypan exclusion. Each sample contained approximately 5×106 total cells, however, viability varied significantly between the three samples. A summary of the cells received and their work-up is shown in Table 3.

TABLE 3# of CellsAllelesPre-Test Sample #CategoryReceivedViabilitySequenced0121-3981-2HLA-A5 × 106N.D.0121-2817-9HLA-B5 × 106N.D.0121-5337-5HLA-C5 × 106>90%Cw*0802,*020220104-8698-32a; HLA-A5 × 106>90%N.D.0104-9545-52b; HLA-B5 × 106>90%B*1510,*5001

[0077] As can be seen from Table 3, samples 3981-2 and 2817-9 were not viable.

[0078] Sample 0121-5337-5 was sequenced according to the method hereinabove described and it was dete...

example 3

[0079] Samples 1548 through 1579 (as shown in Table 4) were received as frozen transformed lymphocytes excepting samples 1551, 1552 and 1553 which were delivered as granulocytes. Once these samples were assigned their respective run numbers processing began. DNA extraction using the QIAMP 96 Spin Blood kit was carried out yielding sufficiently good quality DNA at a suitable concentration for primary PCR amplification. 25 μl primary PCR reactions were set up and were checked by loading 5 μl of the amplicon on a 2% agarose gel run at 200 volts for twenty minutes.

[0080] Once it was established that the primary PCR had produced a suitable product this was then diluted 1:200 with distilled water ready for the secondary PCR reactions. Each dilution was then used in the four secondary PCR reactions with the resulting amplicons again being checked on a 2% agarose gel. The secondary PCR products then underwent solid phase sequencing reactions using the Amersham Pharmacia Autoload SPS kit an...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Volumeaaaaaaaaaa
Volumeaaaaaaaaaa
Volumeaaaaaaaaaa
Login to View More

Abstract

There is provided a method for directly typing or sequencing HLA-A, -B, or -C alleles from a tissue sample wherein exons 2 and 3 of the HLA-A, -B, or -C alleles from the sample are amplified together in a locus specific manner and then separated out and individually amplified in a locus specific manner. After the two amplifications, the amplified exons are directly sequenced, the sequences are recombined, and a comparison is made between the derived HLA allele sequence and an HLA allele database, thereby giving an exact HLA-A, -B, or -C type for the sample being tested.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 10 / 632,571, filed Jul. 28, 2003, which is a continuation of U.S. Ser. No. 09 / 846,826, filed May 1, 2001; which is a continuation of U.S. Ser. No. 09 / 021,892, filed Feb. 11, 1998, now U.S. Pat. No. 6,287,764, issued Sep. 11, 2001; which claims the benefit of U.S. Provisional application Ser. No. 60 / 037,054, filed Feb. 11, 1997.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was developed through assistance with the National Marrow Donor Program (NMDP, contract number 7105) and the Department of Defense, Office of Naval Research (grant number N00014-95-9974).BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates in general to a method for direct DNA sequencing of HLA-A, -B, and -C alleles and more particularly the invention entails the sequence based typing of exons 2 and 3 for the HLA allele gene under study. [0005] 2. Brief D...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12P19/34C12N15/12C12Q1/6881
CPCC12Q1/6881C12Q2600/156
Inventor HILDEBRAND, WILLIAM H.ELLEXSON, MARYCHRETLEN, PIERREDUTHIE, R. SCOTT
Owner HILDEBRAND WILLIAM H
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com