Quantification of polymerase chain reaction products by affinity-based collection

Ann-Christine Syvänen, Hans Söderlund

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

Abstract

To quantify the amount of specific polymerase chain reaction (PCR) product, it is usually not sufficient to measure the total amount of DNA synthesized in the reaction. Depending on the conditions and experimental design, the PCR can create various amounts of product that do not correspond to the desired template. Such erroneous products may arise by the amplification of sequences by essentially random priming or by the creation of primer–dimer artifacts. Once a polymerization product has been formed, it becomes a true PCR template and amplified in subsequent cycles. Consequently, a qualitative step, such as hybridization with a probe internal to the PCR primers, should be included in the analysis. This chapter discusses the application of the affinity-capture technology for the quantification of mixtures of closely related sequences .In this modification of the procedure, the relative concentration of two sequences differing from each other, for example, by a point mutation is accurately determined. Both sequences behave identically in the amplification and one of them serves as an internal standard for the quantification of the other sequence.
Original languageEnglish
Pages (from-to)474 - 490
Number of pages17
JournalMethods in Enzymology
Volume218
DOIs
Publication statusPublished - 1993
MoE publication typeA1 Journal article-refereed

Fingerprint

Polymerase chain reaction
Reaction products
Polymerase Chain Reaction
Amplification
Point Mutation
Polymerization
Design of experiments
Artifacts
Research Design
Technology
DNA

Cite this

Syvänen, Ann-Christine ; Söderlund, Hans. / Quantification of polymerase chain reaction products by affinity-based collection. In: Methods in Enzymology. 1993 ; Vol. 218. pp. 474 - 490.
@article{77db63ba5e7a46bcb2f878ee511fa97a,
title = "Quantification of polymerase chain reaction products by affinity-based collection",
abstract = "To quantify the amount of specific polymerase chain reaction (PCR) product, it is usually not sufficient to measure the total amount of DNA synthesized in the reaction. Depending on the conditions and experimental design, the PCR can create various amounts of product that do not correspond to the desired template. Such erroneous products may arise by the amplification of sequences by essentially random priming or by the creation of primer–dimer artifacts. Once a polymerization product has been formed, it becomes a true PCR template and amplified in subsequent cycles. Consequently, a qualitative step, such as hybridization with a probe internal to the PCR primers, should be included in the analysis. This chapter discusses the application of the affinity-capture technology for the quantification of mixtures of closely related sequences .In this modification of the procedure, the relative concentration of two sequences differing from each other, for example, by a point mutation is accurately determined. Both sequences behave identically in the amplification and one of them serves as an internal standard for the quantification of the other sequence.",
author = "Ann-Christine Syv{\"a}nen and Hans S{\"o}derlund",
note = "Project code: -",
year = "1993",
doi = "10.1016/0076-6879(93)18036-C",
language = "English",
volume = "218",
pages = "474 -- 490",
journal = "Methods in Enzymology",
issn = "0076-6879",
publisher = "Academic Press",

}

Quantification of polymerase chain reaction products by affinity-based collection. / Syvänen, Ann-Christine; Söderlund, Hans.

In: Methods in Enzymology, Vol. 218, 1993, p. 474 - 490.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Quantification of polymerase chain reaction products by affinity-based collection

AU - Syvänen, Ann-Christine

AU - Söderlund, Hans

N1 - Project code: -

PY - 1993

Y1 - 1993

N2 - To quantify the amount of specific polymerase chain reaction (PCR) product, it is usually not sufficient to measure the total amount of DNA synthesized in the reaction. Depending on the conditions and experimental design, the PCR can create various amounts of product that do not correspond to the desired template. Such erroneous products may arise by the amplification of sequences by essentially random priming or by the creation of primer–dimer artifacts. Once a polymerization product has been formed, it becomes a true PCR template and amplified in subsequent cycles. Consequently, a qualitative step, such as hybridization with a probe internal to the PCR primers, should be included in the analysis. This chapter discusses the application of the affinity-capture technology for the quantification of mixtures of closely related sequences .In this modification of the procedure, the relative concentration of two sequences differing from each other, for example, by a point mutation is accurately determined. Both sequences behave identically in the amplification and one of them serves as an internal standard for the quantification of the other sequence.

AB - To quantify the amount of specific polymerase chain reaction (PCR) product, it is usually not sufficient to measure the total amount of DNA synthesized in the reaction. Depending on the conditions and experimental design, the PCR can create various amounts of product that do not correspond to the desired template. Such erroneous products may arise by the amplification of sequences by essentially random priming or by the creation of primer–dimer artifacts. Once a polymerization product has been formed, it becomes a true PCR template and amplified in subsequent cycles. Consequently, a qualitative step, such as hybridization with a probe internal to the PCR primers, should be included in the analysis. This chapter discusses the application of the affinity-capture technology for the quantification of mixtures of closely related sequences .In this modification of the procedure, the relative concentration of two sequences differing from each other, for example, by a point mutation is accurately determined. Both sequences behave identically in the amplification and one of them serves as an internal standard for the quantification of the other sequence.

U2 - 10.1016/0076-6879(93)18036-C

DO - 10.1016/0076-6879(93)18036-C

M3 - Article

VL - 218

SP - 474

EP - 490

JO - Methods in Enzymology

JF - Methods in Enzymology

SN - 0076-6879

ER -