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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

(A) PRIMEGENS-v2 result showing primer sequences, location of the primer pairs with respect to the target gene, amplicon/product size, number of hybridizations and target gene accession numbers for PM18 (Glyma03g34680). (B) Megablast results for forward (A and C) and reverse (B and D) primers in primer pair-1 and primer pair-2 of PM18.
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Figure 8: (A) PRIMEGENS-v2 result showing primer sequences, location of the primer pairs with respect to the target gene, amplicon/product size, number of hybridizations and target gene accession numbers for PM18 (Glyma03g34680). (B) Megablast results for forward (A and C) and reverse (B and D) primers in primer pair-1 and primer pair-2 of PM18.

Mentions: To further validate primer specificity and confirm the capability of PRIMEGENS-v2 to pick a unique region to amplify a specific transcript and a common region to amplify multiple transcripts, we searched these two primer pairs against the Glycine max database. Figure 8A and B shows how PRIMEGENS-v2 precisely picked the unique region of transcript-1 of PM18 (Glyma03g34680) for its unique amplification and common region for the isoforms. The sequence information shows that gene PM18 (Glyma03g34680), located in Gm03: 42 027 662–42 029 006 bp region, has its primary transcript Glyma03g34680.1 that contains one exon between 42 028 575 and 42 029 006 bp in contrast to its alternate transcript Glyma03g34680.2 with the same exon at 42 028 596–42 029 006 bp. On aligning the two primer pairs, we found that PRIMEGENS-v2 picked 20 nt from the exact unique 21 nt region between 42 028 575 and 42 028 795 bp of PM18.1 (Glyma03g34680.1) for primer pair-1 to uniquely amplify PM18.1 and common region for primer pair-2 to pool both isoforms, PM18.1 and PM18.2 (Figure 9).Figure 8.


Homolog-specific PCR primer design for profiling splice variants.

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

(A) PRIMEGENS-v2 result showing primer sequences, location of the primer pairs with respect to the target gene, amplicon/product size, number of hybridizations and target gene accession numbers for PM18 (Glyma03g34680). (B) Megablast results for forward (A and C) and reverse (B and D) primers in primer pair-1 and primer pair-2 of PM18.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: (A) PRIMEGENS-v2 result showing primer sequences, location of the primer pairs with respect to the target gene, amplicon/product size, number of hybridizations and target gene accession numbers for PM18 (Glyma03g34680). (B) Megablast results for forward (A and C) and reverse (B and D) primers in primer pair-1 and primer pair-2 of PM18.
Mentions: To further validate primer specificity and confirm the capability of PRIMEGENS-v2 to pick a unique region to amplify a specific transcript and a common region to amplify multiple transcripts, we searched these two primer pairs against the Glycine max database. Figure 8A and B shows how PRIMEGENS-v2 precisely picked the unique region of transcript-1 of PM18 (Glyma03g34680) for its unique amplification and common region for the isoforms. The sequence information shows that gene PM18 (Glyma03g34680), located in Gm03: 42 027 662–42 029 006 bp region, has its primary transcript Glyma03g34680.1 that contains one exon between 42 028 575 and 42 029 006 bp in contrast to its alternate transcript Glyma03g34680.2 with the same exon at 42 028 596–42 029 006 bp. On aligning the two primer pairs, we found that PRIMEGENS-v2 picked 20 nt from the exact unique 21 nt region between 42 028 575 and 42 028 795 bp of PM18.1 (Glyma03g34680.1) for primer pair-1 to uniquely amplify PM18.1 and common region for primer pair-2 to pool both isoforms, PM18.1 and PM18.2 (Figure 9).Figure 8.

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