Forward And Reverse Primers Pcr

• Journal List
• BMC Biotechnol
• v.14; 2014
• PMC3937175

BMC Biotechnol. 2014; xiv: 10.

Dna polymerase preference determines PCR priming efficiency

Wenjing Pan

¹Biotechnology Scientific discipline and Technology Programme, University of Alabama in Huntsville, Huntsville, AL 35899, USA

Miranda Byrne-Steele

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

Chunlin Wang

³Stanford Genome Engineering science Heart, Stanford University, Palo Alto, CA 94304, Usa

Stanley Lu

⁴Diatherix Laboratories, Huntsville, AL 35806, USA

Scott Clemmons

⁴Diatherix Laboratories, Huntsville, AL 35806, USA

Robert J Zahorchak

ⁱⁱHudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, The states

Jian Han

ⁱⁱHudsonAlpha Plant for Biotechnology, Huntsville, AL 35806, USA

Received 2013 Oct 22; Accepted 2014 Jan 23.

Supplementary Materials

Boosted file 1: Table S1 Barcodes and read coverage are presented. The barcode sequence, the number of full associated reads, the average number of reads per unique sequence in the 4 bp, 6 bp, 8 bp, and ten 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 (equally evidenced by the read distribution) to ensure that both of these libraries had an aplenty number of reads for downstream assay.

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Boosted file ii: Table S2 Statistical assay of compared data sets. A statistical comparing of several data sets is provided with Pearson R, R², p-value, and north.

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Additional file 3: Figure S1 Design for generating unmarried-primer templates and their amplification. (a) The forrad 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 being IgG C-kappa domain. The opposite primer includes the aforementioned filler and 8 bp priming testing site, just includes 18 bp specific for the antisense strand of the kappa domain. After amplification, both the sense and antisense strand comprise the same viii 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 every bit both forward and contrary, consisting of 19 bp of the filler region and 6 bp of the eight bp priming site, is used in the distension experiment.

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Additional file 4 Supplementary Methods.

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Additional file 5: Effigy S2 An example demonstrating the PPI algorithm. Get-go, the 8 bp window is divided into iv 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 sixth position of the viii bp window. The entire 8 bp window is then slid ane bp in the iii' direction, and the process is repeated until the end of the template is reached. A PPI profile of both the sense and antisense strand tin can be generated in order to guide the placement of the 3' stop of the primer into a favorable position.

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Additional file six: Table S3 The importance of the 3' finish of the primer sequence on amplification bias. A detailed sit-in of sequences that have the same GC content just very dissimilar amplification outcomes when amplified with QTT-A DNA polymerase.

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Abstract

Groundwork

Polymerase chain reaction (PCR) is one of the most of import developments in modern biotechnology. However, PCR is known to introduce biases, peculiarly during multiplex reactions. Recent studies have implicated the Deoxyribonucleic acid polymerase every bit the master source of bias, peculiarly initiation of polymerization on the template strand. In our written report, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available Dna polymerases using an anchored primer with a random 3' hexamer stop. Afterwards normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library.

Results

Hither, high throughput sequencing was used to systematically demonstrate and narrate DNA polymerase priming bias. Nosotros demonstrate that sure sequence motifs are preferred over others as primers where the vi nucleotide sequences at the 3' end of the primer, likewise as the sequences 4 base pairs downstream of the priming site, may influence priming efficiencies. Dna polymerases in the same family from two dissimilar commercial vendors adopt similar motifs, while another commercially available enzyme from a unlike Deoxyribonucleic acid polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for sure sequence motifs was verified past amplification from unmarried-primer templates. We incorporated the observed DNA polymerase preference into a primer-blueprint program that guides the placement of the primer to an optimal location on the template.

Conclusions

DNA polymerase priming bias was characterized using a synthetic library amplification organization and NGS. The characterization of Deoxyribonucleic acid polymerase priming bias was and so utilized to guide the primer-design process and demonstrate varying amplification efficiencies amongst three commercially bachelor DNA polymerases. The results propose that the interaction of the DNA polymerase with the primer:template junction during the initiation of Deoxyribonucleic acid polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

Keywords: PCR, Deoxyribonucleic acid polymerase, Priming bias, Next generation sequencing, PPI, Polymerase preference alphabetize, iC-Architect

Background

The polymerase chain reaction (PCR) is one of the near important developments in modern biotechnology. However, PCR is known to introduce biases during amplification, particularly during multiplex PCR when several templates are amplified simultaneously [1,two]. Extreme base compositions (sequences with mostly Yard/C or A/T composition) are recognized to be problematic for both traditional Sanger sequencing and adjacent generation sequencing platforms [3]. However, recent evaluations of biases generated in high throughput sequencing data have pinpointed the amplification stride every bit the primary cause [four,5]. Factors such as thermocycler brand, model, and ramping speed were demonstrated to affect the uniformity of the amplified library [4]. However, the DNA polymerase was identified as the primary source of bias with a variety of commercially bachelor Dna polymerases skewing the amplification profile of the Neandertal genome with regard to GC content and template length [v].

While almost applications focus on or even require the removal of the polymerase-dependent bias, we accept taken an alternative arroyo in which we systematically define the distension bias and utilise information technology to improve PCR success rates. The initiation of DNA polymerization on the template strand is a critical stride in the polymerization process and is likely affected by differences among Dna polymerases and their interaction with the primer:template junction. In support of this notion, a study by Hansen et al. demonstrates that the employ of random hexamer priming induces biases in the nucleotide limerick at the beginning of transcriptome sequencing reads [6]. In our study, nosotros used high throughput sequencing to test the hypothesis that Dna polymerases have a bias for unlike oligonucleotides used every bit primers to initiate DNA synthesis. In society to define this source of Dna polymerase bias, we carefully examined the contribution of the DNA sequence from a ten base pair (bp) window surrounding the primer:template junction including six base of operations pairs (bps) of the primer:template duplex, which rests in the palm of the polymerase prior to nucleotide addition [7,8], and the four bps of single-stranded DNA template immediately following the 90° kink at the junction, which we termed the "runway".

Beginning, we created a constructed sequencing library containing a twelve nucleotide random insertion (12 N) and flanking sequences without the use of amplification. This constructed library (termed SL) provides a library of random sequences that reverberate the complete puddle of template sequences available prior to amplification. The SL was utilized equally the template for several distension experiments. The sequencing results of the distension experiments were compared to the SL, which was sequenced directly (no amplification). Later identifying sequence motifs that were preferentially amplified, single-primer templates were created in guild to verify the DNA polymerase bias. We then developed a primer-design program, iC-Architect, which uses the observed bias in order to improve primer blueprint.

Methods

Constructed library and barcode production

The synthetic library (SL) was produced by the ligation of a barcode segment and a synthesized oligonucleotide (Figure1). The synthesized oligonucleotide contains a 12 North random region, which was converted to a double stranded template equally described beneath. The barcode portion of the SL design allows a unique barcode to be ligated to each of the dissimilar sample amplification test sets so that they can be pooled for high throughput sequencing. The barcode oligo consists of the Illumina adaptor B sequence, a filler region (200 bp long), and a four bp barcode followed by a SfiI site that matches the synthesized oligonucleotide'southward 5' end. The barcode portion was created by PCR distension using a 200 bp portion of the human IgG C-kappa domain as the template to serve as the filler sequence. The filler sequence is used to make the end-product length optimal for span PCR during loftier throughput sequencing. Forward and reverse primers were designed and so that they included the Illumina adaptor B sequence and the barcode (XXXX) with the SfiI ligation site, respectively. The frontward primer utilized is as follows: 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCAGAGAGGCCAAAGTACAG-3', while the reverse primer is 5'-CGTAGGCCACTGAGGCCXXXXTCGCCCCGGTTGAAGCT. Each unique barcode was produced by distension with Qiagen's TopTaq Master Mix Kit in a Bio-Rad C1000 thermocycler as follows: initial denaturation at 94°C for iii minutes; 35 cycles at 94°C for thirty due south, 65°C for 30 s, and 72°C for 40 due south; a terminal extension at 72°C for 10 minutes and so concur at four°C. All amplified products were run on a 1.5% agarose gel, and the gel band of correct size was extracted and purified using Qiagen'southward Qiaquick Gel Extraction Kit every bit per the manufacturer's instructions.

Design of the barcode and constructed random oligonucleotide used to create the SL. The barcode portion was created through PCR distension, while the synthesized random oligonucleotide was purchased and converted to a double stranded template. Both portions were digested with SfiI endonuclease, ligated, gel purified, and sequenced to produce the SL. Exact sequences used to generate the final synthetic library are provided.

The 99 bp synthesized oligo was purchased from IDT with standard desalting. The pattern includes a 17 bp SfiI ligation site, a vii bp spacer, a 12 N random site, an additional 2 bp spacer followed by a 3 N random site to facilitate sequencing, and the Illumina sequencing adaptor A (Figure1). Both the 7 bp and two bp spacer regions adjacent to the 12 N random site were required so that non-specific cleavage of the 12 N region did not occur during digestion with SfiI. The additional three N random site after the two bp spacer is required during sequencing so that the dissimilar clusters can be distinguished during first few cycles of sequencing. The synthesized oligo was converted to a double stranded template using a unmarried reverse primer (5'-AATGATACGGCGACCACCGAGATCT-iii') and 35 cycles of 2 steps of annealing and extension including 55°C for 30 due south and 72°C for 30 s with Qiagen'due south HotStar HiFidelity Deoxyribonucleic acid polymerase. An backlog of cycles was used during this step to ensure that all of the single-stranded oligo was converted to double stranded template. The newly generated double stranded constructed templates were gel purified, and the product was then directly ligated to the barcode portion afterwards SfiI digestion. After ligation, the SL was gel purified after extraction from a 3% agarose gel and sequenced on the Illumina Hiseq 2000 platform using single finish reads from adaptor A through SeqWright's sequencing service. For all subsequent amplification experiments, a unique barcode portion was ligated to the amplified product, gel purified, and sequenced as described. The barcodes associated with a given amplification experiment are summarized in Additional file 1: Tabular array S1.

Amplification experiments

For all distension experiments, ii μL of SL at 50 ng/μL served as the template for amplification, and the same opposite primer as the SL production was used for all amplification experiments. All PCRs were performed as a 50 μL full reaction with 2 μL each of forward and reverse primer at x pmol/μL. All reactions were performed in the buffer system supplied by the respective DNA polymerase vendor without modification. For the amplified background experiment, the forward primer (5'-GCATGGCCTCAGTGGCCTCT-iii') was placed 4 bp upstream of the 12 Northward random site. Distension was performed in a Bio-Rad C1000 thermocycler as follows: initial denaturation at 94°C for three minutes; 35 cycles at 94°C for 30 s, 65°C for thirty s, and 72°C for xxx s; a final extension at 72°C for 10 minutes and with a concur at four°C.

For both the Promega and Qiagen'south Taq DNA polymerases, two repeat amplification experiments were performed for both types of polymerase, while i amplification experiment was performed with Qiagen's HotStar HiFidelity Deoxyribonucleic acid Polymerase. The analysis for only one commercial family unit B DNA polymerase was performed because nosotros needed to ensure that we allotted enough sequencing reads for sufficient coverage of the 12 N random region of all of the currently described distension experiments in 1 Illumina HiSeq sequencing lane. Since the synthetic library is considered a low diversity library, we expected the number of sequencing reads output to be at the lower end of the spectrum. Since we needed to include the different annealing temperatures and repeat experiments for the two Taq Dna polymerases, we limited the number of commercial Dna polymerases in the experiment to three. All polymerases utilized the aforementioned forward primer (5'-GCATGGCCTCAGTGGCCTCTGCATNNNNNN-three') covering 6 nucleotides of the 12 North random portion of the template. All polymerases were subjected to the same cycling atmospheric condition, and amplification was performed as follows: initial denaturation at 94°C for 150 southward; 35 cycles at 94°C for xxx south, lxx–threescore°C touchdown for 120 southward, and 72°C for 30 s; a final extension at 72°C for 10 minutes and then hold at 4°C. For the experiment testing different annealing temperatures, all PCR parameters remained the same with the exception of the annealing temperatures: 63, 65, and 67°C for 100 s.

Statistical analysis of the observed bias value

To assure that the proportion of each unique sequence in the SL was represented in the distension experiments, we counted the reads for each sequence combination for four observation windows (four bp-runway, half-dozen bp-primer:template interaction only, 8 bp- primer:template and runway, and 10 bp-primer:template and runway; Figureii) for barcode 1 (the SL) and designated that result every bit the background for the generated template for all further analyses.

Ascertainment windows for the 12 N portion of the SL. (A) The 4 bp window includes the nucleotides 4 bp ahead of the primer:template junction; (B) the vi bp window includes the primer:template interaction; (C) the 8 bp window includes the primer:template interaction and ii bp alee of the primer:template junction; and (D) the 10 bp window includes the primer:template interaction and four bp alee of the junction.

Since the number of the sequence reads differs between barcoded samples, normalization of the information was required prior to comparison samples. To do so, we adapted the reads from data sets of differing scales to a notionally common scale. A z-score was calculated that adjusted a given sample size to that of the designated background sample (SL), and this standard score process was applied to all ascertainment regions for all barcodes.

To ascertain the degree of bias in a given sample, a relative value for each specific sequence from the ascertainment region was calculated. The normalized reads from the PCR amplified sample (NR_PCR) were divided past the reads for the same sequence from the synthetic library (SR_SL). For instance, for a half dozen bp observation window, assuming the specific DNA sequence "ATCGAT" results in 10 reads from the SL, and the normalized reads from the Taq polymerase PCR sample is 20, the "ATCGAT" at the end of primer has an observed bias value of two. Theoretically, if at that place is no priming bias for the Dna polymerase, the observed bias value for all sequences in different observation regions should be similar to each other and close to a value of "1". The observed bias value (OBV) formula follows:

The statistics for all data comparisons are summarized in Additional file ii: Table S2.

Single-primer exam

Single-primer templates are templates with the same primer binding site on both the sense and anti-sense strands. In total, 24 unique single-primer templates were created and utilized for additional amplification experiments. The unmarried-primer templates were produced past outset designing primers that independent the primer site of interest and a portion of the human being IgG C-kappa domain. Therefore, the forwards primer consisted of a 19 nucleotide filler region, the eight nucleotide primer site to be tested, and nineteen nucleotides specific for the sense strand of the kappa domain, while the contrary primer included the identical filler and primer site with nucleotides specific for the anti-sense strand of the kappa domain (Additional file iii: Figure S1). The kappa domain was amplified as follows: initial denaturation at 94°C for 3 minutes; 35 cycles at 94°C for xxx s, 56°C for 30 southward, and 72°C for 40 southward; a final extension at 72°C for 10 minutes so hold at 4°C. Twenty-four generated templates were separated on 1.v% agarose gel, and the band of the right size was gel purified. All unmarried-primer templates were adapted to the same concentration of 0.001 ng/μL prior to the single-primer distension examination.

For each of the 24 templates, a unique unmarried primer for each template was designed that includes the 19 nucleotide filler region and 6 nucleotides of the eight nucleotide primer site. In each reaction, 2 μL of generated template and ii μL of single primer at x pmol/μL were used with either Qiagen TopTaq or Qiagen HotStar HiFidelity DNA polymerase. End-signal PCR was performed using two annealing temperatures, 57°C and 59°C for 30 s, with either xx or 25 cycles. An internal control for each reaction was also performed with primers specific for an internal portion of the C-kappa region, which was the same for all 24 generated templates (frontwards-5'-TCTGTCTTCATCTTCCCGCCA-3'; contrary-5'AAGCTCTTTGTGACGGGCGAG-3'). Negative control amplification experiments, in which no template was added to the reaction, were performed, and no amplification was observed (data not shown).

Results

Amplification experiments and barcode analysis

In order to define the Deoxyribonucleic acid polymerase bias, we created a synthetic library (SL) of sequences that are identical in sequence with the exception of a 12 nucleotide random insertion (EffigyiiiA). The SL was utilized every bit a template for a variety of amplification experiments, and all amplified libraries, including the non-amplified SL, were pooled and extensively sequenced using the Illumina HiSeq platform. Two primer sets were designed to amplify the SL. The forward and reverse primers of primer set I are located exterior the random-insertion region (Figure3B). The forward primer of primer gear up Ii is located at the purlieus of the random-insertion region with six random nucleotides at the 3' cease of each primer (Figure3C). The contrary primer of primer ready II is identical to the contrary primer of primer set I. The SL was amplified with both primer sets I and Ii using iii commercially available DNA polymerases: two family A Deoxyribonucleic acid polymerases, Qiagen TopTaq (QTT-A) and Promega GoTaq (PGT-A); and one family B DNA polymerase Qiagen HotStar HighFidelity (QHH-B). Repeat amplification experiments were performed at several annealing temperatures (touchdown 70–60°C, 63°C, 65°C, and 67°C) equally described in the Methods.

Experimental prepare-up for the distension of the SL with 2 different primer sets. (A) The SL that contains a 12 Northward random region (blueish) serves as the template for all additional amplification experiments; (B) in the amplified background experiment, the frontwards primer is placed 4 bp upstream of the 12 N region; (C) in the polymerase amplification tests, the forrad primer (grey and pink) covers 6 Due north of the 12 N region.

Prior to data analysis, all high throughput sequencing data were first separated into individual data sets by utilizing the barcodes associated with each experiment, and the data were normalized as described in the Methods. High throughput sequencing of the information sets returned 91,434,220 sequence reads, afterwards filtering past the barcode sequences. The barcode sequences, number of associated reads, and the experimental description matching each barcode are shown in Additional file 1: Table S1. Sequencing of the unamplified SL confirmed that all of the possible combinations of template random nucleotide sequences are represented.

In order to clarify the data relevant to the random region, we filtered the information based on a 4 bp window (runway), 6 bp window (primer:template interaction), eight bp window (primer:template interaction and 2 bps of runway), and 10 bp window (primer:template interaction and 4 bps of runway) equally demonstrated in Figure2. An observed bias value (OBV) was calculated and assigned to each sequence motif in the respective observation window (Methods). Since the 10 bp observation window lacks sufficient read coverage (Additional file ane: Table S1), it was not utilized for further analysis due to lack of statistical significance.

Observation of bias

When primer set I is used to amplify the SL (EffigythreeB), the random sequences are faithfully amplified (Figure4A). However, when a primer with six random bases (6 Due north) at the 3' end is used to amplify the library (Figure3C), priming bias is observed (Figure4B-D). Furthermore, the observed bias is polymerase specific with the two commercially available family A DNA polymerases (QTT-A and PGT-A) preferring a set of sequence motifs unlike from those preferred by the family unit B Dna polymerase (QHH-B; Figure5A-B). For instance, the sequence motif "GGGGGCGG" is the top-ranked one among 65,536 possible motifs for all the QTT-A Deoxyribonucleic acid polymerase distension experiments, including the touchdown, 63°C, 65°C, and 67°C annealing temperatures (Table1). The same sequence motif is ranked second and 3rd, respectively, for PGT-A polymerase. In contrast, this sequence motif is ranked 4,180 for the touchdown experiment of QHH-B. When comparison the top 30 ranked sequence motifs for the QTT-A touchdown PCR experiment across all family A DNA polymerase amplification experiments (including PGT-A), the same 30 sequence motifs announced within the meridian 87 of 65,536 possibilities. In contrast for the QHH-B touchdown experiment, these xxx sequence motifs are ranked from a range of 220 to 29,598 of 65,536 sequences (Tablei).

Bias profiles for the SL and amplification experiments. All bias profiles were created by plotting the observed bias value (OBV) for the four bp (n = 256) and 6 bp (n = iv,096) windows of observation on a logarithmic scale versus the amplified sequences, which were bundled from depression to high GC content in the order of ATCG; (A) the bias observed after amplification with primer prepare I; (B) after distension using primer set II with PGT-A Deoxyribonucleic acid polymerase; (C) using primer prepare Ii with QTT-A Deoxyribonucleic acid polymerase; (D) using primer set II with QHH-B Deoxyribonucleic acid polymerase.

Correlation of amplified libraries. (A) A plot of the OBV of 2 family unit A polymerases (PGT-A versus QTT-A) demonstrates a high caste of correlation throughout the primer:template (6 bp) and runway (4 bp) regions; (B) a plot of the OBV of PGT-A versus OBV of QHH-B indicates that resulting distension profile is weakly correlated for both the primer:template interaction (half-dozen bp) and the rails (4 bp) for 2 differing families of polymerases.

Table one

A comparison of the top thirty ranked QTT-A sequences across all distension experiments

8 bp window sequence	Top xxx ranked QTT-A sequences: comparison of rank in other amplification experiments
	QTT-A	QTT-AR	PGT-A	PGT-AR	QTT-A63	QTT-A65	QTT-A67	QHH-B
GGGGGC GG	i	3	2	3	1	1	1	4180
TTGGGC GG	2	one	three	9	viii	5	2	20382
GGGCCG GG	3	12	5	2	5	three	three	606
TGGCCG GG	4	5	4	iv	6	six	six	3065
GGTCCG GG	v	half-dozen	i	1	three	2	4	1666
GGGTGC GG	6	ix	vi	thirteen	16	12	nine	2377
GTGGGC GG	7	11	10	5	9	7	11	1464
TTGCCG GG	8	8	8	seven	22	21	twenty	13440
GTGCCG GG	9	13	7	viii	7	xiii	17	220
GGGGGC CG	10	19	21	26	two	4	five	3950
TGGTGC GG	11	eighteen	9	14	37	28	19	8530
TTGGGC CG	12	10	19	24	21	27	14	26404
TGCCCG GG	13	14	13	vi	13	fifteen	21	3370
GGTCCG GC	fourteen	17	xi	12	18	18	23	3802
GGGGGC CA	xv	31	24	29	12	14	15	8559
GGGCCG GC	16	32	30	15	10	nine	28	2255
TGGCCG GC	17	xv	xv	16	26	23	22	5312
TTGTGC GG	18	27	23	40	51	69	44	17564
TGTCCG GG	19	21	12	11	32	33	33	9598
TTGGGC CA	20	sixteen	34	35	58	51	47	29598
GGTGGC GG	21	22	16	17	nineteen	22	24	1890
GGGGGC CC	22	36	47	45	fifteen	17	13	10961
TTTGGC GG	23	33	32	22	57	71	39	24345
GGCCCG GG	24	26	14	10	17	xvi	18	508
TGGGCG GG	25	29	29	23	29	47	40	5616
GGGCGC GG	26	87	33	38	25	34	42	1337
TTGCCG GC	27	23	31	32	46	52	65	18324
TTGGGC GC	28	24	51	57	92	62	60	25137
GGGGGC GC	29	47	58	64	27	24	27	9051
GGGGGC TG	30	35	55	eighty	4	8	10	8558

The top 30 sequences of the QTT-A touchdown PCR experiments were ranked and compared to the QTT-A repeat experiment (QTT-AR), the PGT-A and PGT-A repeat experiments (PGT-AR), the QTT-A experiments performed at 63°C, 65°C, and 67°C (QTT-A63, QTT-A65, and QTT-A67), and the QHH-B touchdown PCR amplification experiment.

There is positive amplification bias towards increasing GC content for the six base pairs of primer:template interaction for all of the commercial Dna polymerases tested. This is evident when the OBV for each sequence is plotted with the sequences bundled in order of increasing GC content (Figure4B-D). For the top 1% of ranked sequences, the average GC content is 79% for the family unit A DNA polymerases and 83% for the QHH-B Dna polymerase. We likewise observed that the two bps located closest to the primer:template junction contained loftier GC content in sequences that were preferentially amplified. For instance, for the top i% of ranked sequences of the 8 bp window, "GC" or "CG" is located at the 3' end of the primer for 88-91% of the sequences across all of the family unit A DNA polymerase amplification experiments (Tabular array2). In contrast, 25% of the tiptop-ranked sequences of QHH-B polymerase contain these sequence motifs at the 3' end of the primer. The trend extends to the last three nucleotides on the three' end of the primer. The sequence motifs "CGC", "CCG", "GCG", "GGC", "TGC", and "TCG" occur at the 3' end of the primer for 87-ninety% of the height i% of ranked sequences for the family A DNA polymerases and 22% for the family B Deoxyribonucleic acid polymerase (Table2). In contrast, QHH-B has a preference for "GC" or "CG" at the 5' end of the 6 N portion of the primer with 89% of the pinnacle 1% of ranked sequences containing this motif, while only 6-12% of top ranked sequences from the family A experiments contain these sequences at their v' terminate. Therefore, the specific location of the GC content contributes to the distension bias throughout the primer:template interaction. Interestingly, two GC-rich motifs at the 3' end of the primer, "AGC" and "ACG", had consistently poor amplification results across all polymerases with only i% of top ranked QTT-A and PGT-A results and four% of top ranked QHH-B sequences containing these motifs.

Table two

The 8 bp window motif analyses for several DNA polymerase experiments

Superlative one% (655 in 65,536)	eight bp window motif analyses for several Deoxyribonucleic acid polymerase experiments
	QTT-A	QTT-AR	PGT-A	PGT-AR	QTT-A63	QTT-A65	QTT-A67	QHH-B
(GC,CG) motif at 3' end of primer	88%	89%	90%	90%	90%	91%	88%	25%
(GC,CG) motif at 5' end of 6 N portion of the primer	8%	vi%	8%	eleven%	12%	10%	8%	89%
(TC) motif at first 2 bp of runway	3%	4%	iii%	3%	four%	4%	4%	19%
(CC) motif at first 2 bp of runway	28%	26%	28%	thirty%	23%	23%	25%	30%
(CGC,CCG,GCG, GGC,TGC,TCG) motif at iii' terminate of primer	87%	88%	88%	89%	90%	90%	87%	22%
(AGC,ACG) motif at three' finish of primer	1%	1%	1%	i%	i%	1%	1%	four%
(ACG) motif at 3' cease of primer	one%	1%	one%	1%	ane%	1%	i%	2%
(AGC) motif at 3' end of primer	0	0	0	0	0	0	0	2%

The motif percentage breakdown for the top 1% of ranked sequences are shown for several PCR experiments: QTT-A touchdown, QTT-AR, PGT-A, PGT-AR, QTT-A63, QTT-A65, QTT-A67, and the QHH-B touchdown PCR amplification experiment.

When the OBV for each runway motif is plotted with the sequences arranged in lodge of increasing GC content, the bias profile does not demonstrate a similar design to the primer:template interaction (Figure4B-D). The sequence motif 3'-"TC"-5' is preferred past QHH-B DNA polymerase in the first two positions afterwards the primer:template junction (on the template sequence) with 19% of the superlative 1% of ranked sequences sharing this motif. In dissimilarity, across all family A polymerase experiments, merely 3-iv% of top ranked sequences incorporate this motif at the get-go of the runway (Tabletwo). All of the DNA polymerases tested demonstrate a preference for three'-"CC"-5' in this position with 23-30% of family A and 30% of the family B top ranked sequences sharing this motif.

In gild to examine the effects of annealing temperature, nosotros repeated distension experiments at three boosted annealing temperatures and compared the results to touchdown PCR experiments (Methods). All results were highly correlated with R² > 0.98 (n = 4,096, p = 0.0000; Figure6), indicating that the annealing temperature over the tested range did not have a pregnant effect on the bias profile for the 6 bp observation window.

Affect of annealing temperature on the amplified library. Amplification was performed at 63, 65, and 67°C, and the sequenced reads were compared to bear on downwards PCR experiments (70-60°C) for the six bp observation window.

Overall, QTT-A and PGT-A Dna polymerases take a similar amplification contour with an R² of 0.98 (due north = 4,096; p = 0.0000) for the primer:template interaction despite the fact that they were ordered from two split up vendors and likely work in different buffer conditions (Figure5A). The similarity betwixt the distension profiles for both the primer:template interaction and the track sequences of these ii DNA polymerases is remarkable. However, we observed a dissimilar bias profile when the QTT-A polymerase was compared to the QHH-B DNA polymerase, a family B enzyme (FigurevB). The bias profiles for the two different families of polymerases have an R² of 0.15 (northward = 4,096, p = 0.0000) for the primer:template interaction and an R² of 0.49 (north = 4,096, p = 0.0000) for the runway sequences. An interaction between the primer and template Deoxyribonucleic acid sequences independent of the polymerase cannot be the only cistron leading to the observed bias; otherwise, all of the Dna polymerases would have amplified the SL similarly. The observed differences in distension profiles are likely due to variation in the proprietary buffer conditions and structural differences between the ii Dna polymerase families.

Consideration of Dna polymerase bias during primer blueprint

Based on the observed frequencies of DNA sequence motifs from the high throughput sequencing experiments, we developed a primer-blueprint program chosen iC-Architect to assist PCR primer blueprint by displaying the observed bias aslope target sequences (Additional file 4: Supplementary Methods). The software models the observed Deoxyribonucleic acid polymerase preference by calculating an index value for a sliding eight base pair window, termed the polymerase preference index or PPI, and assigning the PPI value to the template strand (Boosted file 5: Figure S2). An example of the iC-Builder display for a sample target sequence is provided in Figurevii. The software aids in positioning the iii' terminate of the primer in places where the PPI value of the template is at a maximum. As demonstrated by the correlation coefficients in FigureeightA-B and Additional file 2: Tabular array S2, the PPI accurately models the observed polymerase bias from high throughput sequencing experiments. However, there are a few notable outliers betwixt the predicted and observed bias values. After examining the total-length sequence of these particular primers, significant hairpin secondary structure was predicted through IDT's OligoAnalyzer Tool (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/), and poor distension efficiency was observed during the distension experiments. This underscores the importance of avoiding secondary structural elements such equally hairpins when designing primers, and this additional pace should be taken after considering the position of the primer on the template strand.

Output of the iC-Architect software. A graphical display of the output of the iC-Builder online software for a sample template strand. Maximum positions point the optimal placement for the 3' end of the primer, while minimum positions should be avoided.

Understanding between the modeled and observed bias. The polymerase preference alphabetize (PPI) is highly correlated with the observed bias value (OBV) for both the (A) 6 bp and (B) viii bp windows of observation for each of the DNA polymerase tested.

We tested 24 primers that were predicted to have a range of polymerase preference from poor to proficient equally indicated past the direct observed bias (Methods). In all cases, the observed bias was in agreement with the optimal primer position provided by the iC-Architect software and therefore farther served to validate our software. Since we wanted the amplification to exist attributed to only one primer at a time, we generated single-primer templates. These templates include the aforementioned single-primer binding site on both the forward and reverse strands. We designed the single primer to examine an eight bp window, which includes six bps of the primer:template and two nucleotides of the runway. The amplification profiles demonstrate that the bias both observed and predicted through the software can exist replicated experimentally (Effigy9).

Unmarried primer experiments validating suggested primer-design locations from the iC-Architect software. For each of the 24 single-primer templates, the agarose gel results are shown with calculated PPI and OBV values bundled from poor to good. Ii repeat amplifications were performed at 2 temperatures: 59°C (lanes one and 2) and 57°C (lanes three and 4). All amplifications were stopped afterward xx cycles, except lane ane, which was stopped at 25 cycles to demonstrate that 20 cycles was sufficient to see the difference in amplification. An internal control amplification was likewise performed on a portion of the sequence consistent with all templates. T₁₀₀₀ values for the unmarried-primers were calculated using the IDT OligoAnalyzer Tool.

Nosotros besides selected a subset of primer sequences that showed positive amplification bias with the family A DNA polymerases and negative bias with the family unit B Dna polymerase, and vice versa. We performed the single-primer amplification tests with these two opposing subsets and were able to demonstrate that the 2 families of polymerases preferentially dilate certain sequences, yet fail to amplify others (Figureten). Even though the two families of DNA polymerases have different bias profiles, the iC-Architect software is capable of predicting the best locations for the primer as long as the underlying data used to calculate the index refers to the specific commercially available DNA polymerase utilized in the experiment.

Single primer experiments demonstrating the difference in amplification bias betwixt dissimilar polymerase families. For each of the 24 single-primer templates, a subset of sequences for the QHH-B DNA polymerase that have a PPI of the opposite trend with QTT-A DNA polymerase were selected. For agarose gel results, lane 1 shows the QTT-A results while lane 2 shows the QHH-B results, which has PPI values in reverse club. An internal control amplification was also performed on a portion of the sequence consistent with all templates.

The preferential amplification of certain sequences based on the blazon of Dna polymerase utilized indicates that GC and T_k calculations should not exist the only considerations when designing primers. For example, in the single-primer template amplification experiments, the same single primer was used with two different types of DNA polymerases to dilate an identical template nether the same reaction conditions. Although the primers have the same T_thou and GC content, differences in amplification efficiency were readily evident (Figure10). The differences in amplification efficiency may be related to both polymerase blazon and varying buffer conditions amongst commercial vendors. In other words, we cannot rule out that differing buffer atmospheric condition between the family unit A and family unit B Deoxyribonucleic acid polymerase contribute to the observed departure or if it is actual structural differences between the two families of DNA polymerases (or a combination of both). Furthermore, the arrangement of nucleotides in the primer itself has a big bear upon on PCR success. There are many examples from the current loftier throughput sequencing experiment of sequences that have the same nucleotide content (and therefore, the aforementioned GC content), but vastly different outcomes in terms of amplification success due to the bodily arrangement of the nucleotides in the sequence (Figureeleven and Boosted file 6: Table S3). For example, the sequence CCG TTT has an OBV of 0.48, while the sequence TTT CCG has an OBV of 4.55. This is not to say that T_m and GC content should be ignored during primer blueprint. They should be considered in addition to the DNA polymerase preference for specific sequence motifs.

Importance of the 3' finish of the primer sequence on amplification bias. The OBV for the 6 bp window of (due north = iv,096) was plotted against the OBV of the sequence of the reverse trimer social club. For instance the OBV for the 6-mer "CCG AGG" is plotted against the OBV of "AGG CCG". Although the sequences have the same GC content, the amplification results are not correlated.

Discussion

Through the use of the next generation sequencing technology, the current study evaluated the polymerase preference past straight observing the priming efficiency of all possible hexamer primers. The crystal structure of catalytically agile Bacillus stearothermophilus DNA polymerase crystals indicates that approximately 10 bps of primer:template duplex DNA and four nucleotides of template in front of the primer template junction occupy the Deoxyribonucleic acid binding site of the polymerase during synthesis [7,ix]. In improver, the structure reveals a transition from A to B form Dna in the six bps side by side to the primer:template junction. The tendency of a given nucleotide sequence toward A or B conformation is a primal cistron in many poly peptide-DNA interactions [10,11]. For instance, when cyclic AMP receptor protein binds, the Dna undergoes a B to A like transition, and the bounden is stronger when the central part of the Deoxyribonucleic acid is in the A grade [12]. Coordinating to Deoxyribonucleic acid polymerases, the RNA-DNA heteroduplex transiently formed during transcription [13] and the Dna in the active centers of HIV-ane reverse transcriptase [fourteen] are also in the A grade. It has been suggested that the stabilization of the A grade may be critical for increasing the fidelity of DNA and RNA synthesis [15]. In this study, we observed that the two base of operations pairs located at the primer:template junction independent a high GC content in sequences that were preferentially amplified. Several authors have demonstrated that poly (dG)-poly (dC) undergoes the transition from B form to A from more easily than poly (dA)-poly (dT), which tends to resist the B to A transition [12,sixteen]. Indeed, the Gibb's complimentary energy for the B to A transition for GC rich trimeric motifs is lower than the transition of like AT rich sequence motifs [sixteen]. It is reasonable to consider that a primer:template interaction that has a tendency to be of the A form or a primer:template interaction that can exist more easily conformed to the A class is preferentially jump by the DNA polymerase, ultimately resulting in biased amplification.

The purpose of the current written report was to demonstrate and ascertain DNA polymerase-dependent priming bias. Positive distension bias towards increasing GC content was observed for the 6 base pairs of primer:template interaction for all of the commercial Dna polymerases tested, and several preferentially amplified sequence motifs were identified. Interestingly, Dabney et al. establish that some normally used polymerases strongly bias confronting amplification of endogenous Dna in favor of GC-rich microbial contamination [five]. In their study, Phusion HF and AmpliTaq Golden showed a very pronounced bias towards molecules with >50% GC. Another study by Hansen et al. demonstrated that random hexamer priming during the generation of cDNA induces biases in the commencement of nucleotide sequencing reads. In their study, they attempted to discern whether the high throughput sequencing reads originated from the sense strand past second-strand synthesis (from the Deoxyribonucleic acid polymerase) or the antisense strand by first-strand synthesis (from the reverse transcriptase) [half dozen]. Since both strands displayed a similar bias design, they concluded that the second strand DNA synthesis is likely beingness primed past remaining random hexamers in the solution. They as well noted slight differences in the patterns in the sense and antisense strands which they attributed to dissimilar sequence specificities of the reverse transcriptase and DNA polymerase or the issue of nick priming [half dozen]. Our data is in agreement with their determination and suggests that a bulk of the bias is likely due to priming by the random hexamers and the preference of the DNA polymerase for certain primer:template junctions. The reverse transcriptase may also accept preferential sequence motifs as the RNA-DNA heteroduplex in the active middle of HIV-ane opposite transcriptase [14] is of the A grade; nonetheless, reverse transcriptase bias was not addressed in the currently described experiments.

In our study, the four base pairs of single-stranded DNA template immediately following the junction, which we termed the "runway", were also of interest because the polymerase can direct interact with the exposed bases of the single stranded template. This interaction tin can impact amplification. For case, in archaeal family B DNA polymerases, the DNA polymerase possesses a read-ahead office in which polymerization will stall if an uracil is encountered four base pairs alee of the primer:template junction [17,eighteen]. This is not observed with family A Dna polymerases. The runway sequences in the described experiments did not take a clearly defined trend with regards to GC content like the primer:template interaction. Notwithstanding, some caste of bias was evident, and we were able to identify sequence motifs that were preferentially amplified. This suggests that when amplifying targets with universal primers, every bit in the example of certain multiplex PCR reactions, care should be taken to make sure that the primer-template junction is similar for all targets. If the primer:template junction varies alee of the junction, this may result in the DNA polymerase preferentially amplifying "favorable" primer:template junctions over others.

In this work, all experiments were performed using the manufacturers' polymerase-buffer systems directly. Modifying buffer conditions may influence enzyme and/or Deoxyribonucleic acid configuration and attain a successful distension; however, this post-primer design effort may exist avoided past adjusting the primer position slightly during the initial design phase. This is particularly useful when designing primers for multiplex reactions where inter-loci balancing is necessary to reduce conflicts amid primers and targets. During the design of primers for multiplex reactions, the iC-Architect software can be used to select forward and reverse primers of relatively high and like PPI values for multiple targets or to avoid regions with very low PPI values. Ultimately, the software identifies primers with preferred motifs on the 3' end of the primer, which may be used to increase PCR success rates, especially when used in conjunction with T_thou measurements.

Conclusions

Random hexamer priming bias was analyzed past comparing the amplified products of a synthetic library to the unamplified synthetic library. Three commercially bachelor DNA polymerases were utilized to amplify the library, and two of the DNA polymerases (both Taq) demonstrated remarkably similar amplification profiles, peculiarly when compared to the third DNA polymerase of a different family. Preferentially amplified sequence motifs at the three' end of the primer were identified. These motifs demonstrated a marked GC-rich bias blueprint. The identified bias patterns were used to guide primer pattern. Current primer design methods assume that all sequences can be used equally as priming sites, and melting temperature (T_m) and GC measurements are the almost important predictors of priming efficiency. However, our study demonstrates that the template sequence alee of the primer:template junction and the 3' end of the primer can take an effect on the amplification efficiency and should exist considered in add-on to T_m and GC measurements. In the future, we plan to extend the analysis to different commercially available Dna polymerases, specially family B type DNA polymerases. In improver, it would be interesting to examine the effect of common PCR additives on the amplification bias profile and place additives that may reduce bias. Use of the PPI software is available through the iC-Architect site at http://ic-builder.com/. After logging in, users tin can submit a template sequence. A downloadable csv file is generated, which provides the PPI profile for the template, and optimal primer positions are suggested. These results tin can be utilized together with T_k and GC calculations to guide the placement of a primer to preferential locations on the template.

Abbreviations

PCR: Polymerase chain reaction; bp: base pair; bps: Base pairs; SL: Synthetic library; OBV: Observed bias value; QTT-A: Qiagen TopTaq Family A; PGT-A: Promega GoTaq Family A; QHH-B: Qiagen HotStar HighFidelity Family B; PPI: Polymerase preference index; IC: Internal control; NGS: Next generation sequencing; Tm: Melting temperature.

Competing interests

Funding for project materials was provided by iCubate Inc. The authors declare fiscal competing interests for several of the authors listed. Jian Han is the main scientific officer (CSO) and a shareholder for iCubate, Inc., while Stanley Lu and Scott Clemmons perform contract work for iCubate Inc. iCubate Inc. uses the iC-Architect software to pattern multiplex PCR panels for its open up-platform diagnostic panels.

Authors' contributions

WP devised, executed all described experiments, performed data analysis, and contributed significantly to the writing of the manuscript. MBS aided with experimental design, data analysis and provided significant contribution to the writing of the manuscript. CW helped with data analysis and manuscript preparation. SL and SC developed the PPI software. RZ aided with experimental pattern and editing the manuscript. JH supervised the experimental design and execution and contributed to the writing of the manuscript. All authors read and canonical the last manuscript.

Supplementary Material

Additional file 1: Table S1:

Barcodes and read coverage are presented. The barcode sequence, the number of full associated reads, the average number of reads per unique sequence in the 4 bp, six bp, 8 bp, and 10 bp windows, and the DNA polymerase used are provided for each of the pooled distension experiments. During pooling, more of the SL and amplified groundwork were included in the puddle (equally evidenced past the read distribution) to ensure that both of these libraries had an aplenty number of reads for downstream assay.

Additional file 2: Table S2:

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

Additional file 3: Figure S1:

Pattern for generating single-primer templates and their amplification. (a) The forward primer consists of a filler sequence, an 8 bp primer testing window and xviii nucleotides specific for a portion of the sense strand of the human IgG C-kappa domain. The contrary primer includes the aforementioned filler and eight bp priming testing site, merely includes 18 bp specific for the antisense strand of the kappa domain. Subsequently amplification, both the sense and antisense strand contain the same viii bp sequence for unmarried-primer testing. (b) After the templates are generated, gel purified, and the concentration normalized over all samples, a single primer, which serves equally both frontward and opposite, consisting of xix bp of the filler region and half-dozen bp of the 8 bp priming site, is used in the amplification experiment.

Additional file 4:

Supplementary Methods.

Boosted file 5: Effigy S2:

An example demonstrating the PPI algorithm. First, the 8 bp window is divided into four sections: one dimeric scale (DiSc) and iii 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 viii bp window. The unabridged eight bp window is then slid i 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 exist generated in order to guide the placement of the 3' stop of the primer into a favorable position.

Additional file half dozen: Table S3:

The importance of the three' stop of the primer sequence on amplification bias. A detailed demonstration of sequences that take the same GC content only very different amplification outcomes when amplified with QTT-A Deoxyribonucleic acid polymerase.

Acknowledgements

We would like to thank Dr. Preti Jain for her initial input into this project and Jessica Alleyne and Dr. Chris Gunter for reading the original manuscript and providing insightful suggestions for comeback. We would besides like to thank Dr. Ritalinda Lee for her aid with the statistical analysis and manuscript editing.

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Forward And Reverse Primers Pcr,

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937175/

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