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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

Restriction for PCR amplification. Of the four scenarios, (A) alone will produce an amplicon, and only if the interprimer distance is less than the maximum amplicon size cutoff as determined by the user (default 10 000 nt).
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Figure 2: Restriction for PCR amplification. Of the four scenarios, (A) alone will produce an amplicon, and only if the interprimer distance is less than the maximum amplicon size cutoff as determined by the user (default 10 000 nt).

Mentions: As expected in highly redundant genomes like soybean, we observe hits of primer oligos of a forward primer functioning as a reverse primer in some other region due to gene duplication, inversion events or both. In other words, forward primer can act as reverse primer and vice versa, and amplify a completely different gene. Such Megablast hits further complicate the objective of finding specific primer pair for the target sequence and result in cross-amplification. Therefore, once primer pair design using Primer3 is accomplished and Megablast hits for each 3′-end unique oligo from both forward and reverse primers are recorded, PRIMEGENS-v2 pairs forward and reverse primers to check if they can produce potential amplicon by applying the e-PCR (33) algorithm for hybridization restriction. It first checks the occurrence of Megablast hit of any reverse primer near each Megablast hit for each forward primer on the same chromosome. Then, it checks the order and orientation of primer binding, which is essential to successfully amplify a sequence. In addition, even if the primer pair binds in correct order and orientation, the amplicon size should fall within the desired criterion. For example, a primer pair that shows cross-hybridization with amplification size of more than 10 000 nt other than the target region can safely be ignored, as it is not amplifiable. Figure 2 shows an example of primer oligo Megablast hit, which results in successful or failed amplification in PCR. As shown, out of four different cases of primer pair hits, only one case results in successful amplification. Furthermore, it amplifies only if the amplicon size is less than the maximum possible amplicon size (10,000 nt as the default in PRIMEGENS-v2) for RT–PCR.Figure 2.


Homolog-specific PCR primer design for profiling splice variants.

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

Restriction for PCR amplification. Of the four scenarios, (A) alone will produce an amplicon, and only if the interprimer distance is less than the maximum amplicon size cutoff as determined by the user (default 10 000 nt).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Restriction for PCR amplification. Of the four scenarios, (A) alone will produce an amplicon, and only if the interprimer distance is less than the maximum amplicon size cutoff as determined by the user (default 10 000 nt).
Mentions: As expected in highly redundant genomes like soybean, we observe hits of primer oligos of a forward primer functioning as a reverse primer in some other region due to gene duplication, inversion events or both. In other words, forward primer can act as reverse primer and vice versa, and amplify a completely different gene. Such Megablast hits further complicate the objective of finding specific primer pair for the target sequence and result in cross-amplification. Therefore, once primer pair design using Primer3 is accomplished and Megablast hits for each 3′-end unique oligo from both forward and reverse primers are recorded, PRIMEGENS-v2 pairs forward and reverse primers to check if they can produce potential amplicon by applying the e-PCR (33) algorithm for hybridization restriction. It first checks the occurrence of Megablast hit of any reverse primer near each Megablast hit for each forward primer on the same chromosome. Then, it checks the order and orientation of primer binding, which is essential to successfully amplify a sequence. In addition, even if the primer pair binds in correct order and orientation, the amplicon size should fall within the desired criterion. For example, a primer pair that shows cross-hybridization with amplification size of more than 10 000 nt other than the target region can safely be ignored, as it is not amplifiable. Figure 2 shows an example of primer oligo Megablast hit, which results in successful or failed amplification in PCR. As shown, out of four different cases of primer pair hits, only one case results in successful amplification. Furthermore, it amplifies only if the amplicon size is less than the maximum possible amplicon size (10,000 nt as the default in PRIMEGENS-v2) for RT–PCR.Figure 2.

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