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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 all amplified products after qRT-PCR of transcripts in the LEA gene family of soybean. RNA was extracted from seedlings 10 h after ABA stress.
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Figure 6: Dissociation curves for all amplified products after qRT-PCR of transcripts in the LEA gene family of soybean. RNA was extracted from seedlings 10 h after ABA stress.

Mentions: To experimentally validate the manually verified primers designed by PRIMEGENS-v2, qRT-PCR reactions using cDNA samples were conducted as described above. Figure 5 shows the basic flow chart of primer design, verification and validation steps. Isoforms identified were further tested using two sets of primers to uniquely amplify the isoform in one well and to amplify both the reference transcript and its isoform together in another well. In other words, the primer pair in the second well hybridized to a common region in the two sequences. This allowed us to compare the stringency of the primers by distinguishing the two forms rationally in the same PCR reaction. Though PRIMEGENS-v2 is capable of designing primer pairs common to more than two isoforms, we chose to amplify a maximum of two in a single reaction for ease of comparison and to avoid primer limitation. Figure 6 shows the dissociation curve for all amplified products of the LEA gene family in the soybean genome under the ABA stress condition using primers designed by PRIMEGENS-v2. A curve showing a single dominant peak represents unique product amplification. The profiling results show that while some primers designed by PRIMEGENS-v2 were able to amplify the isoforms together, some others could uniquely amplify each variant and homolog separately, as intended.Figure 5.


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 all amplified products after qRT-PCR of transcripts in the LEA gene family of soybean. RNA was extracted from seedlings 10 h after ABA stress.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105385&req=5

Figure 6: Dissociation curves for all amplified products after qRT-PCR of transcripts in the LEA gene family of soybean. RNA was extracted from seedlings 10 h after ABA stress.
Mentions: To experimentally validate the manually verified primers designed by PRIMEGENS-v2, qRT-PCR reactions using cDNA samples were conducted as described above. Figure 5 shows the basic flow chart of primer design, verification and validation steps. Isoforms identified were further tested using two sets of primers to uniquely amplify the isoform in one well and to amplify both the reference transcript and its isoform together in another well. In other words, the primer pair in the second well hybridized to a common region in the two sequences. This allowed us to compare the stringency of the primers by distinguishing the two forms rationally in the same PCR reaction. Though PRIMEGENS-v2 is capable of designing primer pairs common to more than two isoforms, we chose to amplify a maximum of two in a single reaction for ease of comparison and to avoid primer limitation. Figure 6 shows the dissociation curve for all amplified products of the LEA gene family in the soybean genome under the ABA stress condition using primers designed by PRIMEGENS-v2. A curve showing a single dominant peak represents unique product amplification. The profiling results show that while some primers designed by PRIMEGENS-v2 were able to amplify the isoforms together, some others could uniquely amplify each variant and homolog separately, as intended.Figure 5.

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