Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease

Charul Gijavanekar, Ulrich Strych, Yuriy Fofanov, George E. Fox, Richard C. Willson

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.

Original languageEnglish (US)
Pages (from-to)81-85
Number of pages5
JournalAnalytical Biochemistry
Volume421
Issue number1
DOIs
StatePublished - Feb 1 2012
Externally publishedYes

Fingerprint

Polymerase chain reaction
Nucleic Acids
Polymerase Chain Reaction
Pathogens
Amplification
Sensitivity and Specificity
DNA

Keywords

  • Cot analysis
  • Cot-rehybridization
  • Duplex-specific nuclease
  • PCR diagnostics
  • PCR sensitivity
  • Target enrichment

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease. / Gijavanekar, Charul; Strych, Ulrich; Fofanov, Yuriy; Fox, George E.; Willson, Richard C.

In: Analytical Biochemistry, Vol. 421, No. 1, 01.02.2012, p. 81-85.

Research output: Contribution to journalArticle

Gijavanekar, Charul ; Strych, Ulrich ; Fofanov, Yuriy ; Fox, George E. ; Willson, Richard C. / Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease. In: Analytical Biochemistry. 2012 ; Vol. 421, No. 1. pp. 81-85.
@article{6a3f3d4daa9749cdb38093a46a9492b1,
title = "Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease",
abstract = "Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.",
keywords = "Cot analysis, Cot-rehybridization, Duplex-specific nuclease, PCR diagnostics, PCR sensitivity, Target enrichment",
author = "Charul Gijavanekar and Ulrich Strych and Yuriy Fofanov and Fox, {George E.} and Willson, {Richard C.}",
year = "2012",
month = "2",
day = "1",
doi = "10.1016/j.ab.2011.11.010",
language = "English (US)",
volume = "421",
pages = "81--85",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease

AU - Gijavanekar, Charul

AU - Strych, Ulrich

AU - Fofanov, Yuriy

AU - Fox, George E.

AU - Willson, Richard C.

PY - 2012/2/1

Y1 - 2012/2/1

N2 - Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.

AB - Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.

KW - Cot analysis

KW - Cot-rehybridization

KW - Duplex-specific nuclease

KW - PCR diagnostics

KW - PCR sensitivity

KW - Target enrichment

UR - http://www.scopus.com/inward/record.url?scp=84855877011&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84855877011&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2011.11.010

DO - 10.1016/j.ab.2011.11.010

M3 - Article

VL - 421

SP - 81

EP - 85

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -