Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase
The Stoffel fragment of Taq DNA polymerase lacks the 5′to 3′exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We therefore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a seque...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
1997-10-01
|
| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/97234st06 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850152527449817088 |
|---|---|
| author | Dan Yu Masaya Mukai Qingli Liu Charles R. Steinman |
| author_facet | Dan Yu Masaya Mukai Qingli Liu Charles R. Steinman |
| author_sort | Dan Yu |
| collection | DOAJ |
| description | The Stoffel fragment of Taq DNA polymerase lacks the 5′to 3′exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We therefore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDNA of three bacterial species: E. coli, Bacillus subtilis and Neisseria gonorrhoea. A single pair of primers was capable of amplifying a homologous 240-bp region from all three. Two nonextendable “blocking” oligonucleotides were synthesized with sequences complementary to the inter-primer regions of E. coli and B. subtilis, respectively. Both blockers were shown specifically to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the non-complementary N. gonorrhea. Specificity was further confirmed by an internal positive control. Similar inhibition was seen with mixtures of targets in a single reaction. With intact Taq DNA polymerase, no blocking was observed. Primers and blockers targeting specific regions of N. gonorrhoea rDNA were used to confirm the requirement that blockers be directed to the inter-primer region. Sequence-dependent amplification inhibition, such as that demonstrated here, would be applicable to PCR-related strategies using primers capable of using multiple targets, where such selective inhibition could be useful. |
| format | Article |
| id | doaj-art-1c409085ae9c4e27977ebde1f98a2f86 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 1997-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-1c409085ae9c4e27977ebde1f98a2f862025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98181997-10-0123471472010.2144/97234st06Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA PolymeraseDan Yu0Masaya Mukai1Qingli Liu2Charles R. Steinman31State University of New York at Stony Brook, Stony Brook, NY, USA1State University of New York at Stony Brook, Stony Brook, NY, USA1State University of New York at Stony Brook, Stony Brook, NY, USA1State University of New York at Stony Brook, Stony Brook, NY, USAThe Stoffel fragment of Taq DNA polymerase lacks the 5′to 3′exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We therefore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDNA of three bacterial species: E. coli, Bacillus subtilis and Neisseria gonorrhoea. A single pair of primers was capable of amplifying a homologous 240-bp region from all three. Two nonextendable “blocking” oligonucleotides were synthesized with sequences complementary to the inter-primer regions of E. coli and B. subtilis, respectively. Both blockers were shown specifically to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the non-complementary N. gonorrhea. Specificity was further confirmed by an internal positive control. Similar inhibition was seen with mixtures of targets in a single reaction. With intact Taq DNA polymerase, no blocking was observed. Primers and blockers targeting specific regions of N. gonorrhoea rDNA were used to confirm the requirement that blockers be directed to the inter-primer region. Sequence-dependent amplification inhibition, such as that demonstrated here, would be applicable to PCR-related strategies using primers capable of using multiple targets, where such selective inhibition could be useful.https://www.future-science.com/doi/10.2144/97234st06 |
| spellingShingle | Dan Yu Masaya Mukai Qingli Liu Charles R. Steinman Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase BioTechniques |
| title | Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase |
| title_full | Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase |
| title_fullStr | Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase |
| title_full_unstemmed | Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase |
| title_short | Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase |
| title_sort | specific inhibition of pcr by non extendable oligonucleotides using a 5 to 3 exonuclease deficient dna polymerase |
| url | https://www.future-science.com/doi/10.2144/97234st06 |
| work_keys_str_mv | AT danyu specificinhibitionofpcrbynonextendableoligonucleotidesusinga5to3exonucleasedeficientdnapolymerase AT masayamukai specificinhibitionofpcrbynonextendableoligonucleotidesusinga5to3exonucleasedeficientdnapolymerase AT qingliliu specificinhibitionofpcrbynonextendableoligonucleotidesusinga5to3exonucleasedeficientdnapolymerase AT charlesrsteinman specificinhibitionofpcrbynonextendableoligonucleotidesusinga5to3exonucleasedeficientdnapolymerase |