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Sequence-Specific Oligonucleotide (SSO) Probes

Introduction

Sequence-Specific Oligonucleotide (SSO) probes

are used in many important hybridization techniques including DNA microarrays designed for large-scale variation analysis and genotyping of complex DNA samples.

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome and play a critical but as yet largely uncharacterized role in human disease. Rough estimates suggest that there are 10-30 million SNPs in the human genome, or 1 about every 100-300 bases, on average.

Millions of human SNPs have been catalogued and made available in public repositories such as the NCBI dbSNP and International HapMap Project (HapMap).

Brief review of SNP discovery and genotyping techniques can be found here.

Below is description of SSO hybridization-based SNP genotyping approach used in Affymetrix GeneChip assay.

How It Works

SNP-containing regions are amplified from genomic DNA, cleaved, tagged, and hybridized to the probe array under stringent conditions, with washing followed by fluorescent labeling.

Fragment selection by PCR for Affymetrix Mapping Assay

Affymetrix microarray design

Probes (25-mers) are synthesized as perfect matches (PM) and as one-base mismatches (MM). A perfect match for each of the two SNP alleles (A, B) and one mismatch for each of the two alleles with the nucleotide in question at a given position are referred to as a (probe quartet). There are a total of 56 probes for a SNP miniblock: seven probe quartets containing one SNP at seven different positions along both strands of the sequence. It is possible to reduce the number of probes per SNP from 56 to 40 without loss of accuracy.

Note: only perfectly matched probes were deposited in the NCBI Probe database. In some cases, either sense or antisense probes with the SNP at central, 0th (13th) position are present on the probe report page, and the rest of the probes are omitted.

Terminology and Abbreviations

Ancestry-Informative Markers (AIMs)

  • a subset of SNPs whose allele frequencies differ substantially in one population versus the other two: there are 343,788 and 374 SNPs with FST values >0.4 in the African-American versus Caucasian, African-American versus Asian, and Caucasian versus Asian comparisons, respectively.

Average Call Rate

  • number of SNPs identified, divided by the total number examined.

Fragment Selection by PCR

  • optimization of PCR conditions to selectively amplify DNA fragments of certain size (400 to 800 base pairs (bp)).

F Statistics (FST)

  • a fixation index that compares the average heterozygosity within groups to the total heterozygosity. FST can be used to measure the level differentiation between populations at a locus: 0-0.05, little differentiation; 0.05-0.15, moderate; 0.15-0.25, significant; >0.25, great differentiation.

Minor Allele Frequency (MAF)

  • the ratio of chromosomes in the population carrying the less common SNP to those with the more common SNP.

Relative Allele Signal (RAS)

  • a measure of the proportion of the signal intensities contributed from the A allele compared to signals from the A and B alleles combined; this is calculated for both the sense and antisense strands. Thus, heterozygous genotypes will have a RAS value approaching 0.5, whereas homozygous genotypes will have a RAS value of 1 for the A allele and 0 for the B allele.

Linkage Disequilibrium (LD)

  • a non-random association of alleles at two or more loci, not necessarily on the same chromosome.

Whole Genome Sampling Analysis (WGSA)

  • genotyping thousands of SNPs simultaneously in a complex DNA sample.

References

» Kennedy GC, et al. Large-scale genotyping of complex DNA. Nat Biotechnol. 2003 Oct;21(10):1233-7. Epub 2003 Sep 7. PMID: 12960966

» Matsuzaki H, et al. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res. 2004 Mar;14(3):414-25. PMID: 14993208

» Matsuzaki H, et al. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods. 2004 Nov;1(2):109-111. PMID: 15782172

Resources

» "Oligonucleotide Array Sequence Analysis"[MAJR] AND "Variation (Genetics)"[MAJR]

Note: [MAJR] is a Medical Subject Heading (MeSH) tag for Major Heading. The tag is used to limit the search to articles for which major subjects are represented by terms included in the NLM MeSH database.

Affymetrix SSO

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Last updated: 2017-11-09T11:31:42Z