rapid multiplex snp genotyping using biochip array …

RAPID MULTIPLEX SNP GENOTYPING USING BIOCHIP ARRAY TECHNOLOGY

Randox Laboratories Limited, 55 Diamond Road, Crumlin BT29 4QY, United Kingdom

Doherty JE, Latten MJ, Murray HA, Crockard MA, Lamont JV, FitzGerald SP

Single Nucleotide Polymorphisms (SNPs) represent the most common form of sequence variation in human DNA. SNPs are highly abundant, stable and distributed throughout the human genome, and are associated with diversity in the population, susceptibility to disease and individual response to medicine.1

Through Genome Wide Association Studies, large numbers of

SNPs are being identified that can predict a patient’s response to therapy or adverse reaction to the drug action.2 By individualising treatment, one can maximise drug efficacy and minimize adverse patient effects. However as many of these actions are polygenetic, the capability of multiplexing SNPs reliably at different loci is advantageous.3

This study reports the development of a rapid multiplex SNP genotyping system using Biochip Array Technology in order to provide a reliable platform for 8 SNPs to be genotyped in a single PCR reaction.

Introduction

Conclusions

The Randox Biochip array platform can rapidly genotype 8 SNPs in a single PCR reaction. The platform therefore has wide ranging applicability in the field of personalised medicine and can aid the clinician in selecting the right drug for the right patient, to ensure maximum efficacy and minimal adverse effects.

REFERENCES1. Shastry BS. SNP alleles in human disease and evolution. J Hum Genet. 2002; 47(11):561-6.2. Daly AK. Using genome-wide association studies to identify genes important in serious adverse drug reactions. Annu Rev Pharmacol Toxicol. 2012;52:21-353. Johnson JA. Drug target pharmacogenomics: an overview. Am J Pharmacogenomics 2001; 1(4):271-81.

Methodology

Results

MOLECULAR DIAGNOSTICS

Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United KingdomT +44 (0) 28 9442 2413 F +44 (0) 28 9445 2912 E [email protected] I www.randox.com

The rapid multiplex SNP genotyping system is based on innovative primer design which can discriminate DNA sequences which differ only at one base. For detection of each SNP, a generic, labelled forward primer and 2 reverse primers were designed. One of these reverse primers is complementary to the ancestral base, with the other complementary to the variant sequence. Multiplex of 8 SNPs was then achieved in a single PCR reaction.

Products amplified using multiplex PCR correspond to targeted portions of genomic DNA from blood. Amplified regions were then hybridised to a biochip array with spatially tethered

probes complementary to target amplicons. Each position on the biochip array therefore corresponds to a specific SNP genotype. Chemiluminescent signal is added to the biochip and results imaged using the Evidence Investigator analyser (EV3602, Randox Laboratories Limited, Crumlin, UK). This platform is capable of both multiplexing and determining the zygosity of the sample.

The process takes 3 hours from extracted DNA to result readout. Only 10ng of input DNA is required per PCR reaction, with dedicated software processing results automatically.

Overview of rapid multiplex SNP genotyping array protocol

Genotype Ancestral RLU Variant RLUHomozygous Wild type + -

Heterozygous Mutant + +

Homozygous Mutant - +

Assay verification 8 SNPs were fully genotyped using the biochip array platform. 30 pre-characterised DNA samples (Coriell Cell Repositories, Camden, NJ) were used as reference material. The samples were chosen so that all possible SNP genotypes (homozygous wild-type, heterozygous mutant, homozygous mutant) could be evaluated. The concordance between the Coriell data and the genotypes obtained using biochip array technology was 100%.

Sample assessment100 patient samples were then tested with SNPs being readily detected using the biochip system. The data presented corresponds to 3 patient samples as examples.

Zygosity is determined by comparing generated ancestral Relative Light Units (RLU) with the variant RLU

SNP Biochip Array:Images representing 3 patient samples

SNP biochip array layout

Sample 1SNP No. Ancestral RLU Variant RLU Genotype

1 1270 0 Homozygous Wild type


3 1409 1471 Heterozygous Mutant


5 0 2332 Homozygous Mutant




RLU (Relative Light Unit) readouts for patient samples 1-3







6 0 993 Homozygous Mutant












ReferenceSNP 4

A (Ancestral)SNP 3

G (Ancestral)SNP 2

A (Ancestral)SNP 1

C (Ancestral)

SNP 8A (Ancestral)

SNP 7G (Ancestral)

SNP 6A (Ancestral)

SNP 5T (Ancestral)

Reference

SNP 5C (Variant)

SNP 4G (Variant)

SNP 3C (Variant)

SNP 2C (Variant)

SNP 1T (Variant)

SNP 8G (Variant)

SNP 7 A (Variant)

SNP 6G (Variant)

Sample 1 Sample 2 Sample 3

ML-RA-004-12, MLHM-RA-001-12, ML-RA-007-12

rapid multiplex snp genotyping using biochip array …

Documents