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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

Exploitation of small gaps as unique sequences scattered across whole gene body. Upper panel shows primer pairs to uniquely identify two different splice variants with each circled set of primers specific for a splice-variant. Lower panel shows a set of primer pairs that will amplify both splice variants. Reference gene is shown on the top.
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Figure 1: Exploitation of small gaps as unique sequences scattered across whole gene body. Upper panel shows primer pairs to uniquely identify two different splice variants with each circled set of primers specific for a splice-variant. Lower panel shows a set of primer pairs that will amplify both splice variants. Reference gene is shown on the top.

Mentions: In case of closely related sequences like splice variants, some scattered primers (either forward or reverse primer) lie in the variable regions of paralogs/splice variants, which can uniquely amplify individual genes. Figure 1 shows two different cases of splice variants, where uniqueness of a single primer is used to amplify individual genes. In the upper panel of Figure 1, the first case shows the possibility of designing unique forward primer with common reverse primer whereas the second case uses a variable region to design unique reverse primer with common forward primer. This method will thus use small sequence differences among variants for their unique identification. In contrast, the lower panel shows a set of primer pairs that will amplify both splice variants indiscriminately.Figure 1.


Homolog-specific PCR primer design for profiling splice variants.

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

Exploitation of small gaps as unique sequences scattered across whole gene body. Upper panel shows primer pairs to uniquely identify two different splice variants with each circled set of primers specific for a splice-variant. Lower panel shows a set of primer pairs that will amplify both splice variants. Reference gene is shown on the top.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Exploitation of small gaps as unique sequences scattered across whole gene body. Upper panel shows primer pairs to uniquely identify two different splice variants with each circled set of primers specific for a splice-variant. Lower panel shows a set of primer pairs that will amplify both splice variants. Reference gene is shown on the top.
Mentions: In case of closely related sequences like splice variants, some scattered primers (either forward or reverse primer) lie in the variable regions of paralogs/splice variants, which can uniquely amplify individual genes. Figure 1 shows two different cases of splice variants, where uniqueness of a single primer is used to amplify individual genes. In the upper panel of Figure 1, the first case shows the possibility of designing unique forward primer with common reverse primer whereas the second case uses a variable region to design unique reverse primer with common forward primer. This method will thus use small sequence differences among variants for their unique identification. In contrast, the lower panel shows a set of primer pairs that will amplify both splice variants indiscriminately.Figure 1.

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