Question

In our case, we Blasted to see which gene the primers were amplifying, and wanted to predict exactly how many nucleotides would be in the amplicon. But what if we wanted to know something about this gene? You now know Y54E10A is a YAC that has a number of predicted genes. Which gene is predicted that overlaps with our primers?

Answer 1

partially overlapping primer-based PCR for genome walking we have developed a novel SWPOP-PCR method for genome waliking to isolate and identify the unknown DNA sequences flanking the known segments. Compared to the conventional POP-PC SWPOP-PCR has the merits of simplicity and efficacy due to requiring fewer primers and being suitable for making mix master. The SWPOP-PCR is an alternative of the existing genome walking methods Genome walking of the ad locus in L brevis NCL912 and b integrated in rice To confirm the validity of SWPOP-PCR, we employed the method to isolate target segments flanking the regions of ผ A locus in L brevis NCL91 2 and bus integrated in rice four sets of parallel PCR reactions were successfully performed for each DNA sample, respectively. One or more clear predominant DNA band(s) appeared after the secondary and tertiary PCR reactions A flanking region of approximate 1.2- 16 kb in size was uncovered in each walking Sequencing the bands in the tertiary PCRs showed that the obtained sequences were completely overlapped with the ends of the known gene region in some cases, more than one target DNA bands suggested that a SWPOP-P primer annealed to multiple sites of a DNA plate. in our PCR method, a SWPOP-P primer should find an adapted site in the plate given the fact that one super low-stringency (25 *C) cycle was performed in primary PCR To increase the success rate in genomic walking we designed four parallel SWPOP-P primers having heterologous 15 ba at the 3-parts and identical 10 ba at the 5-parts. The four heterologous 3'-parts are expected to guarantee at least one SWPOP-P primer creates an annealing site adapted for this SWPOP-P within genomic DNA Abbreviations SWPOP-POR stepwise partially overlapping primer-based PCR POP-PCR partially overlapping primer-based PCR adA glutamate decarboxylase gene Lbrevis NCL912 Lactobacillus brevis NC1912 SWPOP-P., SWPOP-S, and SWPOP-T stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively SP-P, SP-5, and SP-T specific primer for primary, secondary, and tertiary PCR, respectively