scholarly journals Direct Cloning of PCR Products Amplified with Pwo DNA Polymerase

BioTechniques ◽  
1996 ◽  
Vol 20 (2) ◽  
pp. 186-188 ◽  
Author(s):  
Stefan Hinnisdaels ◽  
Jurgen Del-Favero ◽  
Marc Vauterin
Keyword(s):  
Dna Polymerase ◽  
Direct Cloning ◽  
Pcr Products

scholarly journals Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products

1991 ◽  
Vol 19 (5) ◽  
pp. 1154-1154 ◽  
Author(s):  
Douglas Marchuk ◽  
Mitchell Drumm ◽  
Ann Saulino ◽  
Francis S. Collins
Keyword(s):  
General System ◽  
Direct Cloning ◽  
Pcr Products

scholarly journals Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning

2001 ◽  
Vol 67 (2) ◽  
pp. 880-887 ◽  
Author(s):  
Xiaoyun Qiu ◽  
Liyou Wu ◽  
Heshu Huang ◽  
Patrick E. McDonel ◽  
Anthony V. Palumbo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Rrna Gene ◽  
Community Studies ◽  
Positive Bacterium ◽  
16S Ribosomal Dna ◽  
Model Community ◽  
Pcr Products ◽  
Alpha Beta ◽  
Optimal Approach

ABSTRACT To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteriaas well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three TaqDNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq(8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

scholarly journals A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses

1997 ◽  
Vol 25 (9) ◽  
pp. 1864-1865 ◽  
Author(s):  
T. S. Gritsun ◽  
M. V. Mikhailov ◽  
P. Roy ◽  
E. A. Gould
Keyword(s):  
Simple Procedure ◽  
Direct Cloning ◽  
Pcr Products

New vectors for direct cloning of PCR products

Gene ◽  
1993 ◽  
Vol 136 (1-2) ◽  
pp. 369-370 ◽  
Author(s):  
Jooyeun Cha ◽  
William Bishai ◽  
Srinivasan Chandrasegaran
Keyword(s):  
Direct Cloning ◽  
Pcr Products

Construction of new T vectors for direct cloning of PCR products

Gene ◽  
1993 ◽  
Vol 130 (1) ◽  
pp. 153-154 ◽  
Author(s):  
Yoshikazu Ichihara ◽  
Yoshikazu Kurosawa
Keyword(s):  
Direct Cloning ◽  
Pcr Products

New vectors for direct cloning of PCR products

Gene ◽  
1994 ◽  
Vol 141 (1) ◽  
pp. 149 ◽  
Author(s):  
Jooyeun Cha ◽  
William Bishai ◽  
Srinivasan Chandrasegaran
Keyword(s):  
Direct Cloning ◽  
Pcr Products

Single-step direct cloning of PCR products

1995 ◽  
Vol 11 (1) ◽  
pp. 7-8 ◽  
Author(s):  
Shuang-En Chuang ◽  
Kao-Chung Wang ◽  
Ann-Lii Cheng
Keyword(s):  
Single Step ◽  
Direct Cloning ◽  
Pcr Products

scholarly journals PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample

2005 ◽  
Vol 71 (12) ◽  
pp. 8966-8969 ◽  
Author(s):  
Silvia G. Acinas ◽  
Ramahi Sarma-Rupavtarm ◽  
Vanja Klepac-Ceraj ◽  
Martin F. Polz
Keyword(s):  
16S Rrna ◽  
Dna Polymerase ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Pcr Products ◽  
Diversity Estimates ◽  
Sequence Types ◽  
16S Rrna Clone Libraries

ABSTRACT The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

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