Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization

Yasuhiro Tsubosa, Hiroyuki Sugihara, Ken Ichi Mukaisho, Sumihiro Kamitani, Dunfa Peng, Zhi Qiang Ling, Tohru Tani, Takanori Hattori

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.

Original languageEnglish (US)
Pages (from-to)156-166
Number of pages11
JournalCancer Genetics and Cytogenetics
Volume158
Issue number2
DOIs
StatePublished - Apr 15 2005
Externally publishedYes

Fingerprint

Comparative Genomic Hybridization
Metaphase
Sensitivity and Specificity
Polymerase Chain Reaction
DNA
Chromosomal Instability
Tetraploidy
DNA Probes
Aneuploidy
Chromosomes
ribonucleotide polymerase
Lymphocytes
Genes
Neoplasms

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

Cite this

Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization. / Tsubosa, Yasuhiro; Sugihara, Hiroyuki; Mukaisho, Ken Ichi; Kamitani, Sumihiro; Peng, Dunfa; Ling, Zhi Qiang; Tani, Tohru; Hattori, Takanori.

In: Cancer Genetics and Cytogenetics, Vol. 158, No. 2, 15.04.2005, p. 156-166.

Research output: Contribution to journalArticle

Tsubosa, Yasuhiro ; Sugihara, Hiroyuki ; Mukaisho, Ken Ichi ; Kamitani, Sumihiro ; Peng, Dunfa ; Ling, Zhi Qiang ; Tani, Tohru ; Hattori, Takanori. / Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization. In: Cancer Genetics and Cytogenetics. 2005 ; Vol. 158, No. 2. pp. 156-166.
@article{7b95d9c217f04eff9aea02c30c8a6b80,
title = "Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization",
abstract = "Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100{\%}) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.",
author = "Yasuhiro Tsubosa and Hiroyuki Sugihara and Mukaisho, {Ken Ichi} and Sumihiro Kamitani and Dunfa Peng and Ling, {Zhi Qiang} and Tohru Tani and Takanori Hattori",
year = "2005",
month = "4",
day = "15",
doi = "10.1016/j.cancergencyto.2004.08.033",
language = "English (US)",
volume = "158",
pages = "156--166",
journal = "Cancer Genetics and Cytogenetics",
issn = "0165-4608",
publisher = "Elsevier Inc.",
number = "2",

}

TY - JOUR

T1 - Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization

AU - Tsubosa, Yasuhiro

AU - Sugihara, Hiroyuki

AU - Mukaisho, Ken Ichi

AU - Kamitani, Sumihiro

AU - Peng, Dunfa

AU - Ling, Zhi Qiang

AU - Tani, Tohru

AU - Hattori, Takanori

PY - 2005/4/15

Y1 - 2005/4/15

N2 - Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.

AB - Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.

UR - http://www.scopus.com/inward/record.url?scp=15744392236&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=15744392236&partnerID=8YFLogxK

U2 - 10.1016/j.cancergencyto.2004.08.033

DO - 10.1016/j.cancergencyto.2004.08.033

M3 - Article

C2 - 15796963

AN - SCOPUS:15744392236

VL - 158

SP - 156

EP - 166

JO - Cancer Genetics and Cytogenetics

JF - Cancer Genetics and Cytogenetics

SN - 0165-4608

IS - 2

ER -