 Flowable demineralized bone powder composition and its use in bone repair |
| What is claimed is: 1. A flowable composition for application to a bone defect site to promote new ... |
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| Crosslinkable polypeptide compositions |
| OF THE INVENTION In accordance with the present invention, there are provided chemically modified ... |
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DNA sequence-based HLA typing method
| Details |
Inventors: Santamaria, Pedro; Boyce-Jacino, Michael Thomas; Barbosa, Jose Joaquim; Rich, Stephen Saul; Faras, Anthony James;
Assignee: Regents Of The University Of Minnesota (Minneapolis, MN)
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Tung; Joyce
Attorney, Agent or Firm: Rae-Venter Law Group, P.C.
The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of CDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed. |
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DETAILED DESCRIPTION The present invention relates to a method for determining the nucleic acid sequence of one or more polymorphic genes of a subject by amplifying and direct sequencing genomic or complementary DNA molecules for each allele at each gene locus to be sequenced using conserved and non-conserved (non-allele-specific) oligonucleotide primers.
In a broad sense, the method of the present invention involves sequence-based typing (SBT) which provides for unequivocal determination of genetic polymorphism at any genetic locus of interest by direct, simultaneous, sequence analysis of both genomic DNA or expressed (RNA) copies of such a locus.
SBT can be employed to determine genetic polymorphism at one or more genetic loci of interest, regardless of the complexity of the polymorphism at these loci, including, for example: (1) simple homozygosity or heterozygosity of a unique locus, as exemplified by DQA or the like; (2) isotypic complexity due to multiple, closely related and closely linked copies of a locus, as exemplified by DRB or the like; and (3) intra-allelic complexity at a locus compounded by interlocus complexity, such as Class I genes or the like.
Most known human genetic polymorphisms are of the first, and simplest, type.
Use of the SBT method provides overlapping sequence data comprised of only the copies of the locus of interest as is exemplified by each of the types of HLA loci.
The SBT strategy is designed to ensure selection of a given locus with equal representation of each copy of that locus by equal amplification and direct sequencing of mixtures of both alleles of that locus and direct interpretation of the overlapping sequencing patterns generated by this approach.
Thus, providing a method for determining genetic polymorphism at one or more genetic loci of interest which can be employed, for example, in HLA typing, detection, evaluation, and/or characterization of genetic diseases such as, for example, sickle cell anemia, cystic fibrosis, Thalassemia, and the like, and detection, evaluation, and/or characterization of polymorphism in genetic loci associated with various cancers such as p53, Ras, myc, associated with carcinomas, leukemias, sarcomas or the like
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