Open Access Highly Accessed Research article

DNA polymerase preference determines PCR priming efficiency

Wenjing Pan1, Miranda Byrne-Steele2, Chunlin Wang3, Stanley Lu4, Scott Clemmons4, Robert J Zahorchak2 and Jian Han2*

Author Affiliations

1 Biotechnology Science and Engineering Program, University of Alabama in Huntsville, Huntsville, AL 35899, USA

2 HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA

3 Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA

4 Diatherix Laboratories, Huntsville, AL 35806, USA

For all author emails, please log on.

BMC Biotechnology 2014, 14:10  doi:10.1186/1472-6750-14-10

Published: 30 January 2014

Additional files

Additional file 1: Table S1:

Barcodes and read coverage are presented. The barcode sequence, the number of total associated reads, the average number of reads per unique sequence in the 4 bp, 6 bp, 8 bp, and 10 bp windows, and the DNA polymerase used are provided for each of the pooled amplification experiments. During pooling, more of the SL and amplified background were included in the pool (as evidenced by the read distribution) to ensure that both of these libraries had an ample number of reads for downstream analysis.

Format: PDF Size: 65KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2: Table S2:

Statistical analysis of compared data sets. A statistical comparison of several data sets is provided with Pearson R, R2, p-value, and n.

Format: PDF Size: 75KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 3: Figure S1:

Design for generating single-primer templates and their amplification. (a) The forward primer consists of a filler sequence, an 8 bp primer testing window and 18 nucleotides specific for a portion of the sense strand of the human IgG C-kappa domain. The reverse primer includes the same filler and 8 bp priming testing site, but includes 18 bp specific for the antisense strand of the kappa domain. After amplification, both the sense and antisense strand contain the same 8 bp sequence for single-primer testing. (b) After the templates are generated, gel purified, and the concentration normalized over all samples, a single primer, which serves as both forward and reverse, consisting of 19 bp of the filler region and 6 bp of the 8 bp priming site, is used in the amplification experiment.

Format: PDF Size: 167KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 4:

Supplementary Methods.

Format: PDF Size: 71KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 5: Figure S2:

An example demonstrating the PPI algorithm. First, the 8 bp window is divided into four sections: 1 dimeric scale (DiSc) and three trimeric scales (TrSc). The PPI value for each of the scales is based on the nucleotide sequence of the dimer or trimer and its relative position in the 8 bp window. The product of the DiSc and three TrScs is calculated and assigned to the 6th position of the 8 bp window. The entire 8 bp window is then slid 1 bp in the 3’ direction, and the process is repeated until the end of the template is reached. A PPI profile of both the sense and antisense strand can be generated in order to guide the placement of the 3’ end of the primer into a favorable position.

Format: PDF Size: 854KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 6: Table S3:

The importance of the 3’ end of the primer sequence on amplification bias. A detailed demonstration of sequences that have the same GC content but very different amplification outcomes when amplified with QTT-A DNA polymerase.

Format: PDF Size: 76KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data