A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

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Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719866484 ◽

2019 ◽

Vol 31 (5) ◽

pp. 714-718 ◽

Cited By ~ 2

Author(s):

Kristin A. Clothier ◽

Simone Stoute ◽

Andrea Torain ◽

Beate Crossley

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Limit Of Detection ◽

Bacterial Flora ◽

Clinical Samples ◽

Single Tube ◽

Avibacterium Paragallinarum ◽

Normal Bacterial Flora ◽

Close Relatives

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89–111%. Cross-reaction was not detected with 33 non– A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

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High‐Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems

Current Protocols in Molecular Biology ◽

10.1002/0471142727.mb0705s96 ◽

2011 ◽

Vol 96 (1) ◽

Cited By ~ 18

Author(s):

Derek Daigle ◽

Birgitte B. Simen ◽

Pascale Pochart

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Life Sciences ◽

Universal Primers ◽

Pcr Products

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Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽

10.1139/g99-120 ◽

2000 ◽

Vol 43 (2) ◽

pp. 412-415 ◽

Cited By ~ 4

Author(s):

Zhong-Nan Yang ◽

T Erik Mirkov

Keyword(s):

Restriction Enzyme ◽

Genomic Dna ◽

Enzyme Digestion ◽

Restriction Enzyme Digestion ◽

Artificial Chromosomes ◽

Bac Clones ◽

Anchored Pcr ◽

Pcr Products ◽

Left And Right ◽

The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

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Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase (UNG) in a single tube reaction

Journal of Clinical Laboratory Analysis ◽

10.1002/(sici)1098-2825(1997)11:6<323::aid-jcla2>3.0.co;2-6 ◽

1997 ◽

Vol 11 (6) ◽

pp. 323-327 ◽

Cited By ~ 10

Author(s):

Saibal K. Poddar ◽

Mark H. Sawyer ◽

James D. Connor

Keyword(s):

Influenza A Virus ◽

Dna Polymerase ◽

Influenza A ◽

Pcr Amplification ◽

Single Tube ◽

Virus Rna ◽

Single Tube Reaction

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.880-887.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 880-887 ◽

Cited By ~ 291

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.

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