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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

Distribution of 15-mer primer oligos based on their Megablast hits in the Glycine max database. The peak at hit count = 500 combines all the occurrences with hits of 500 or more.
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Figure 4: Distribution of 15-mer primer oligos based on their Megablast hits in the Glycine max database. The peak at hit count = 500 combines all the occurrences with hits of 500 or more.

Mentions: In order to estimate the complexity of finding sequence-specific primer pairs, we randomly chose approximately 26 000 15-mer oligos derived from more than 100 000 oligos designed as primer for various exonic regions from the soybean genome. The uniqueness of these 15-mer oligos was checked against the Glycine max database, which contains 77 778 sequences, using Megablast. Figure 4 shows the distribution of oligo hit counts on the Glycine max database, where vast majority of oligos show more than two hits, with only seven out of 26 331 hitting once and only 15 giving two hits. The peak in this plot corresponds to oligos that give 30–40 hits on the Glycine max database. The large number of hits is mainly due to genome complexity of soybean. Soybean genome is a product of an ancient genome duplication event and multiple neo-polyploid speciation events imposed on its paleopolyploid genome. In addition, gene loss, gene silencing and rearrangement of the genome like inversion or translocations along with duplication events (41,42) further made the genome more complex and gave rise to unusually high number of similar sequence fragments within its genome.Figure 4.


Homolog-specific PCR primer design for profiling splice variants.

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

Distribution of 15-mer primer oligos based on their Megablast hits in the Glycine max database. The peak at hit count = 500 combines all the occurrences with hits of 500 or more.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Distribution of 15-mer primer oligos based on their Megablast hits in the Glycine max database. The peak at hit count = 500 combines all the occurrences with hits of 500 or more.
Mentions: In order to estimate the complexity of finding sequence-specific primer pairs, we randomly chose approximately 26 000 15-mer oligos derived from more than 100 000 oligos designed as primer for various exonic regions from the soybean genome. The uniqueness of these 15-mer oligos was checked against the Glycine max database, which contains 77 778 sequences, using Megablast. Figure 4 shows the distribution of oligo hit counts on the Glycine max database, where vast majority of oligos show more than two hits, with only seven out of 26 331 hitting once and only 15 giving two hits. The peak in this plot corresponds to oligos that give 30–40 hits on the Glycine max database. The large number of hits is mainly due to genome complexity of soybean. Soybean genome is a product of an ancient genome duplication event and multiple neo-polyploid speciation events imposed on its paleopolyploid genome. In addition, gene loss, gene silencing and rearrangement of the genome like inversion or translocations along with duplication events (41,42) further made the genome more complex and gave rise to unusually high number of similar sequence fragments within its genome.Figure 4.

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