Detection of mutations in insulin receptor gene by denaturing gradient gel electrophoresis. Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5' end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied.

# When designing primers how important is the GC clamp? Tags: pcr, primer - Score: 11 - Views: 25676 - Answers: 4 - Answered: yes - Asked by: Ben (1131 rep) - Asked: 2013-10-13 - Site: biology ## Question I'm designing a set of primers and reading about the principles of primer design one of which is: GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer. From here. My question is how essential is it to have a GC clamp? ## Answers ### Answer by user4675 (score: 10 [ACCEPTED]) It's hard to provide an objective answer. If you have a decent length and good complexity, even a single terminal 3' G or C would do. Of course, one has to take into account the primer's overall GC:AT ratio and things like annealing temperature. Here's a link to diverse opinions on the topic and it has this nugget (which I subscribe to when possible): FWIW, my preferred offerings to the PCR gods are primers with a single G or C 3', FWIW. Seems to keep 'em happy most

his chapter discusses a trend in 3′-end triplet frequencies in primers used in successful PCR experiments and proposes requirements for the 3′-end of a primer. Finally, a method break to select primers with the best 3′-end triplets is introduced based on the 3′-end analysis result. Key Words: PCR, primer design, 3′-end, VirOligo, primers, viruses, oligonucleotides Introduction The - and -ends of a primer have different meanings for PCR processes. Complementarity of the -end of a primer to the PCR template is not so critical as for the -end, and it is known that longer primers at the -end (such as 30 nt or longer) do not improve specificity of PCR primers. The -end of a primer also allows an addition of a tagging sequence. Complete binding between primers and template is not required at the -end. However, the -end of a primer is different from the -end. Thermostable DNA polymerase starts attaching nucleotides from the -end of a primer during the extension step, and it requires complete annealing of the -end of a primer to a template. Incomplete binding at the -end results in inefficient PCR or sometimes no PCR products. Alternatively, it is possible that too stable annealing of a primer at the -end to a template allows generation of PCR product without complete binding between the rest of primer and template, and tolerance in incomplete binding may amplify unexpected PCR product by primer binding to other templates or different regions in a target template. Thus, the -end of a primer is important in PCR primer design for successful PCR experiments. The -End of a Primer Recommended in Literature Several kinds of recommendations for the -end of a primer can be found in the literature. One recommends one or two S (S stands for C or G) at the -end triplet of a primer for promoting strong annealing at the -end ( 1 , 2 ). Another also recommends C or G at the -end of a primer but no CG or GC due to potential formation of hairpins and primer-dimers ( 3 ). Although incorpora

Technical report: Part 2. Basic requirements for designing optimal PCR primers ## Abstract Designing optimal polymerase chain reaction (PCR) primer sequences is one of the critical factors for successful PCR with sensitive, specific, and assay-to-assay reproducible results. In this review, all the requirements of PCR primer sequences are summarized, such as location, size of amplicon, length of primers, nucleotide composition, Tm, 3' terminal hybridization strength and frequency, hairpin formation energy, primer-to-primer interaction, specificity, and location of mismatches to sequences of cross-hybridization. The report also discusses how to explore these various types of information for more advanced PCR applications, which include nested PCR, multiplex PCR, competitive PCR, long PCR, point mutation detection, degenerate primers, and PCR cloning. ## MeSH terms - DNA Primers* - Nucleic Acid Hybridization - Point Mutation - Polymerase Chain Reaction* - Sensitivity and Specificity ## Substances - DNA Primers