Polymerase chain reaction primer design

Regarding reverse transcriptase polymerase chain reaction (RT-PCR) analysis to assess a splice site variant, as seen in Fig. 12.6, if one designs forward (F-) and reverse (R-) RT-PCR primers to span the SNP (which in turn creates or abolishes a splice site), one wiU have different size PCR products (labeled a, b, and c in the figure) that can easily be resolved on a gel. 

A set of sense and antisense primers should be selected to synthesize specific cDNAs and also to detect the amplified messages of the genes, complementary to their specific gene sequences. It is important to consider the following points while designing the primers for reverse transcriptase and polymerase chain reaction ... 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules. 

Fig. 2. Direct transcriptional template generation from cDNA library by using the split-primers polymerase chain reaction (PCR) technique. (a-c) A schematic representation of the split-primers design for equipping the cDNA sequences with required UTRs, where b and c are expected PCR-generated DNAs and mRNA, respectively, (cl) Split-primer PCR-generated products.

Fig. 5. (Opposite page) Polymerase chain reaction (PCR)-based expression of cDNA information using conventional method (A) and a new strategy using split-type primers (B). a. Design of the split-type primers for the introduction of the required UTRs into cDNA sequences, b and c. Expected PCR-generated DNAs and mRNA, respectively, d. PCR-generated DNA. e. A 10-pL aliquot from aPCR sample was used forthe 100-pL transcription reaction, and transcripts were analyzed, f. All of the transcript was used for batch-mode translation (50 pL, 4 h), and products were analyzed by autoradiography. (From ref. 36a.)...

Marks JD, Tristem, Karpas A, Winter G, Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes, Eur. J. Immunol., 21 985-991, 1991. 

Bingle et al. (3) realized that these subtle protein sequence differences should be reflected in DNA sequence, and polymerase chain reaction (PCR) primers could be designed to... 

The solid-phase synthesis of DNA oligomers has reached a high level of maturity and is extensively used for the synthesis of primers for polymerase chain reaction or the design of new genes (13). In contrast to the cellular process, the synthetic route forms the polymer in 3 -to-5 direction. The oligomerization is carried out on CPG using a base labile linker. Typically, the nucleobases are introduced as acyl-protected phosphoramidites,... 

Polymerase chain reaction is particularly useful for genetic analysis because both amplification and primer specific isolation of gene fragments occur simultaneously. Allele-specific amplification can be employed to detect a single base-pair mutation through the use of a specially designed primer which is complementary to... 

Fransen,K Zhong, P., De Beenhouwer, H Carpels, G Peeters, M., Louwagie, J., Janssens, W., Piot, P., and van der Groen, G. (1995) Design and evaluation of new, highly sensitive and specific primers for polymerase chain reaction detection of HIV-1 infected primary lymphocytes. Mol. Cell. Probes 9, 373. 

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