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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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Basic computational steps in PRIMEGENS-v2 for homolog-specific primer design (HSPD).
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Figure 3: Basic computational steps in PRIMEGENS-v2 for homolog-specific primer design (HSPD).

Mentions: After this step, the primer pair pool generated gets further reduced to retain only those primer pairs that result in potentially good amplicons with no or few cross-hybridization. Out of these final candidates, the top primer pair(s) having no or the least amplifiable primer-pair products in the whole genome is reported as the final output. Figure 3 shows the basic flowchart of major computational steps. Primer pair combinations can then be manually verified and selected based on the target gene attributes such as splice variants and duplicates. The design provides detailed information about all primers such as their start and end positions on query sequence; number, names and sizes of all potential amplicons; and melting temperatures and GC contents of both forward and reverse primers. Manual verification adds another layer of confidence but the user has the option of by passing this and instead selecting top primer pairs provided by PRIMGENS-v2. Selection can also be made based on the design purpose. For example, whether an isoform has to be uniquely amplified or a pool of isoforms needs to be amplified.Figure 3.


Homolog-specific PCR primer design for profiling splice variants.

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

Basic computational steps in PRIMEGENS-v2 for homolog-specific primer design (HSPD).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Basic computational steps in PRIMEGENS-v2 for homolog-specific primer design (HSPD).
Mentions: After this step, the primer pair pool generated gets further reduced to retain only those primer pairs that result in potentially good amplicons with no or few cross-hybridization. Out of these final candidates, the top primer pair(s) having no or the least amplifiable primer-pair products in the whole genome is reported as the final output. Figure 3 shows the basic flowchart of major computational steps. Primer pair combinations can then be manually verified and selected based on the target gene attributes such as splice variants and duplicates. The design provides detailed information about all primers such as their start and end positions on query sequence; number, names and sizes of all potential amplicons; and melting temperatures and GC contents of both forward and reverse primers. Manual verification adds another layer of confidence but the user has the option of by passing this and instead selecting top primer pairs provided by PRIMGENS-v2. Selection can also be made based on the design purpose. For example, whether an isoform has to be uniquely amplified or a pool of isoforms needs to be amplified.Figure 3.

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