Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization

H. van Dekken, D. Pinkel, J. Mullikin, Joe Gray

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.

Original languageEnglish (US)
Pages (from-to)1-5
Number of pages5
JournalChromosoma
Volume97
Issue number1
DOIs
StatePublished - Sep 1988
Externally publishedYes

Fingerprint

Single-Stranded DNA
Fluorescence In Situ Hybridization
Human Chromosomes
Digestion
Chromosomes
Fluorescence
Deoxyribonuclease EcoRI
Chromosomes, Human, Pair 1
Telomere
Biotin
Acetic Acid
In Situ Hybridization
Methanol
DNA
exodeoxyribonuclease III

ASJC Scopus subject areas

  • Genetics

Cite this

Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization. / van Dekken, H.; Pinkel, D.; Mullikin, J.; Gray, Joe.

In: Chromosoma, Vol. 97, No. 1, 09.1988, p. 1-5.

Research output: Contribution to journalArticle

van Dekken, H. ; Pinkel, D. ; Mullikin, J. ; Gray, Joe. / Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization. In: Chromosoma. 1988 ; Vol. 97, No. 1. pp. 1-5.
@article{786bd211be8f4b60be61786d9a0a4f02,
title = "Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization",
abstract = "This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.",
author = "{van Dekken}, H. and D. Pinkel and J. Mullikin and Joe Gray",
year = "1988",
month = "9",
doi = "10.1007/BF00331788",
language = "English (US)",
volume = "97",
pages = "1--5",
journal = "Chromosoma",
issn = "0009-5915",
publisher = "Springer Science and Business Media Deutschland GmbH",
number = "1",

}

TY - JOUR

T1 - Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization

AU - van Dekken, H.

AU - Pinkel, D.

AU - Mullikin, J.

AU - Gray, Joe

PY - 1988/9

Y1 - 1988/9

N2 - This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.

AB - This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.

UR - http://www.scopus.com/inward/record.url?scp=0023693656&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023693656&partnerID=8YFLogxK

U2 - 10.1007/BF00331788

DO - 10.1007/BF00331788

M3 - Article

C2 - 3191791

AN - SCOPUS:0023693656

VL - 97

SP - 1

EP - 5

JO - Chromosoma

JF - Chromosoma

SN - 0009-5915

IS - 1

ER -