Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

Claus Børsting; Juan J Sanchez; Niels Morling

doi:10.1007/s00414-003-0419-y

Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

Claus Børsting, Juan J Sanchez, Niels Morling

Section of Forensic Genetics

15 Citations (Scopus)

Abstract

We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

Original language	English
Journal	International Journal of Legal Medicine (Print)
Volume	118
Issue number	2
Pages (from-to)	75-82
Number of pages	7
ISSN	0937-9827
DOIs	https://doi.org/10.1007/s00414-003-0419-y
Publication status	Published - 2004

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10.1007/s00414-003-0419-y

Cite this

@article{ba694b6097ae11de8bc9000ea68e967b,

title = "Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray",

abstract = "We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.",

author = "Claus B{\o}rsting and Sanchez, {Juan J} and Niels Morling",

note = "Keywords: Chromosomes, Human, Y; DNA Fingerprinting; Humans; Male; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Sensitivity and Specificity",

year = "2004",

doi = "10.1007/s00414-003-0419-y",

language = "English",

volume = "118",

pages = "75--82",

journal = "International Journal of Legal Medicine",

issn = "0937-9827",

publisher = "Springer",

number = "2",

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TY - JOUR

T1 - Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

AU - Børsting, Claus

AU - Sanchez, Juan J

AU - Morling, Niels

N1 - Keywords: Chromosomes, Human, Y; DNA Fingerprinting; Humans; Male; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Sensitivity and Specificity

PY - 2004

Y1 - 2004

N2 - We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

AB - We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

U2 - 10.1007/s00414-003-0419-y

DO - 10.1007/s00414-003-0419-y

M3 - Journal article

C2 - 14722709

SN - 0937-9827

VL - 118

SP - 75

EP - 82

JO - International Journal of Legal Medicine

JF - International Journal of Legal Medicine

IS - 2

ER -