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Next-generation Sequencing

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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RNA-seq

RNA-seq

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qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

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Published on: May 25, 2015

KIR genotyping by reverse sequence-specific oligonucleotide methodology.

T Nong¹, K Saito, L Blair

¹One Lambda, Inc., Canoga Park, CA 91303-2801, USA.

Tissue Antigens

|April 21, 2007

Summary

Killer cell immunoglobulin-like receptor (KIR) genotyping is crucial for stem cell transplantation. A new bead-based assay using Luminex technology accurately identifies KIR genes and alleles, including complex variants.

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Area of Science:

Immunogenetics
Molecular Biology
Transplantation Science

Background:

Killer cell immunoglobulin-like receptors (KIR) play a role in immune regulation.
Accurate KIR genotyping is essential for optimizing bone marrow and stem cell transplantation outcomes.
Existing genotyping methods may face challenges with certain KIR gene polymorphisms.

Purpose of the Study:

To develop a novel bead-based assay for Killer cell immunoglobulin-like receptor (KIR) genotyping.
To utilize the Luminex platform for high-throughput and precise KIR DNA analysis.
To enable accurate identification of KIR genes, pseudogenes, and null alleles.

Main Methods:

Development of a bead-based reverse sequence-specific oligonucleotide (SSO) DNA hybridization assay.
Design of oligonucleotide probes targeting 14 known KIR genes, 2 pseudogenes, and null alleles.
Implementation of a unique probe strategy for KIR2DS4 cis-polymorphic region detection.
Validation using DNA samples from the 13th International Histocompatibility Working Group.

Main Results:

The developed assay successfully genotyped 14 KIR genes, 2 pseudogenes, and null alleles.
A novel probe design enabled accurate typing of the KIR2DS4 gene, including its polymorphic regions.
Assay performance demonstrated 100% concordance with established typing methods from the 13th International Histocompatibility Working Group.

Conclusions:

The bead-based Luminex SSO assay provides a robust and accurate method for KIR genotyping.
This assay facilitates precise identification of KIR alleles relevant to stem cell transplantation.
The method offers improved detection capabilities for complex KIR polymorphisms, enhancing its clinical utility.