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Homolog-specific PCR primer design for profiling splice variants.

Srivastava GP, Hanumappa M, Kushwaha G, Nguyen HT, Xu D - Nucleic Acids Res. (2011)

Bottom Line: Results indicate a high success rate of primer design.Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene.Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family.

View Article: PubMed Central - PubMed

Affiliation: Department of Computer Science, Christopher S Bond Life Sciences Center, University of Missouri and Informatics Institute, University of Missouri, Columbia, MO 65211, USA. gsrivastava@rics.bwh.harvard.edu

ABSTRACT
To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.

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Related in: MedlinePlus

Dissociation curves for PM18 transcript in the control and ABA-treated samples showing primer specificity. Red and green curves represent amplicons generated by two different sets of primers. (A) PM18.2 is abundant in the control condition (green arrow). The small, red and green peaks in the dashed box indicate comparatively low abundance of PM18.1. (B) Both PM18.1 and PM18.2 are expressed in the ABA-treated samples. Curves represent amplicons derived from primer pairs designed to amplify PM18.1 (red arrow) alone in well B2 or both PM18.1 (dotted green arrow) and PM18.2 (solid green arrow) in well B3. The similarity of PM18.1 peaks within (A) or within (B) indicates primer stringency.
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Figure 7: Dissociation curves for PM18 transcript in the control and ABA-treated samples showing primer specificity. Red and green curves represent amplicons generated by two different sets of primers. (A) PM18.2 is abundant in the control condition (green arrow). The small, red and green peaks in the dashed box indicate comparatively low abundance of PM18.1. (B) Both PM18.1 and PM18.2 are expressed in the ABA-treated samples. Curves represent amplicons derived from primer pairs designed to amplify PM18.1 (red arrow) alone in well B2 or both PM18.1 (dotted green arrow) and PM18.2 (solid green arrow) in well B3. The similarity of PM18.1 peaks within (A) or within (B) indicates primer stringency.

Mentions: In this article, we present primers designed to amplify PM18 (Glyma03g34680), a member of the LEA family, as evidence for primer stringency, accuracy and specificity, as designed by PRIMEGENS-v2. Figure 7 shows two sets of primer pairs represented in green and red for PM18 gene. One of these primer pairs (shown in green) loaded in well B3 was capable of amplifying both splice variants of the gene PM18 (PM18.1 and PM18.2; Glyma03g34680.1 and Glyma03g34680.2), while another primer pair (shown in red) in well B2 could uniquely amplify PM18.1. The curves represent two wells that use different primer sets for the two PCR reactions on cDNA synthesized from the control and ABA stressed samples. A smaller peak at a lower Tm in Figure 7A is a result of very low transcript abundance. In Figure 7B, where primers were designed to detect more than one form as represented by the green curve, the peak is still sharp, with a second peak at a lower temperature. This second peak is easily distinguishable from that in Figure 7A due to its height, which is directly related to the abundance of the amplicon. From this, we can conclude that PM18.1 has very low expression in Figure 7A and that its expression is enhanced when the plant is stressed with ABA (as seen in Figure 7B). However, PM18.2 expression in stress is comparable to that in the control. By comparing between Figure 7A and B, we can clearly rule out primer dimerization as a result of low transcript abundance in Figure 7A. Taken together, the curves genuinely reflect transcript profiles and hence, primer specificity.Figure 7.


Homolog-specific PCR primer design for profiling splice variants.

Srivastava GP, Hanumappa M, Kushwaha G, Nguyen HT, Xu D - Nucleic Acids Res. (2011)

Dissociation curves for PM18 transcript in the control and ABA-treated samples showing primer specificity. Red and green curves represent amplicons generated by two different sets of primers. (A) PM18.2 is abundant in the control condition (green arrow). The small, red and green peaks in the dashed box indicate comparatively low abundance of PM18.1. (B) Both PM18.1 and PM18.2 are expressed in the ABA-treated samples. Curves represent amplicons derived from primer pairs designed to amplify PM18.1 (red arrow) alone in well B2 or both PM18.1 (dotted green arrow) and PM18.2 (solid green arrow) in well B3. The similarity of PM18.1 peaks within (A) or within (B) indicates primer stringency.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105385&req=5

Figure 7: Dissociation curves for PM18 transcript in the control and ABA-treated samples showing primer specificity. Red and green curves represent amplicons generated by two different sets of primers. (A) PM18.2 is abundant in the control condition (green arrow). The small, red and green peaks in the dashed box indicate comparatively low abundance of PM18.1. (B) Both PM18.1 and PM18.2 are expressed in the ABA-treated samples. Curves represent amplicons derived from primer pairs designed to amplify PM18.1 (red arrow) alone in well B2 or both PM18.1 (dotted green arrow) and PM18.2 (solid green arrow) in well B3. The similarity of PM18.1 peaks within (A) or within (B) indicates primer stringency.
Mentions: In this article, we present primers designed to amplify PM18 (Glyma03g34680), a member of the LEA family, as evidence for primer stringency, accuracy and specificity, as designed by PRIMEGENS-v2. Figure 7 shows two sets of primer pairs represented in green and red for PM18 gene. One of these primer pairs (shown in green) loaded in well B3 was capable of amplifying both splice variants of the gene PM18 (PM18.1 and PM18.2; Glyma03g34680.1 and Glyma03g34680.2), while another primer pair (shown in red) in well B2 could uniquely amplify PM18.1. The curves represent two wells that use different primer sets for the two PCR reactions on cDNA synthesized from the control and ABA stressed samples. A smaller peak at a lower Tm in Figure 7A is a result of very low transcript abundance. In Figure 7B, where primers were designed to detect more than one form as represented by the green curve, the peak is still sharp, with a second peak at a lower temperature. This second peak is easily distinguishable from that in Figure 7A due to its height, which is directly related to the abundance of the amplicon. From this, we can conclude that PM18.1 has very low expression in Figure 7A and that its expression is enhanced when the plant is stressed with ABA (as seen in Figure 7B). However, PM18.2 expression in stress is comparable to that in the control. By comparing between Figure 7A and B, we can clearly rule out primer dimerization as a result of low transcript abundance in Figure 7A. Taken together, the curves genuinely reflect transcript profiles and hence, primer specificity.Figure 7.

Bottom Line: Results indicate a high success rate of primer design.Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene.Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family.

View Article: PubMed Central - PubMed

Affiliation: Department of Computer Science, Christopher S Bond Life Sciences Center, University of Missouri and Informatics Institute, University of Missouri, Columbia, MO 65211, USA. gsrivastava@rics.bwh.harvard.edu

ABSTRACT
To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.

Show MeSH
Related in: MedlinePlus