Background: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability.
Utilizing in vitro transcription and translation (IVTT) to produce small quantities of proteins is convenient but requires a significant supply of pure template DNA. This can be cumbersome, particularly when the method is used for many different templates in a high-throughput manner. Multiply-primed rolling circle amplification (RCA) with [#x03C6]29 DNA polymerase is a simple way to generate large amounts of DNA; however, the products of this amplification method have interruptions in both strands and branched structures. In this study, we tested whether RCA-generated DNA can serve as the template for in vitro transcription. We found that RCA DNA[#x02013]generated transcripts work in coupled in vitro translation with nearly the same efficiency (per nanogram of DNA) as those obtained from purified plasmid. We propose a convenient, single-tube format for template amplification, transcription, and translation.
A series of 5' deletion, internal deletion, and linker-scanning mutants of the minute virus of mice P4 promoter were constructed and analyzed for transcriptional activity in nuclear extracts of mouse A92L fibroblasts. A GC box and a TATA box essential for in vitro transcription from the P4 promoter were localized between nucleotides 150 and 180 (-55 to-25 relative to the primary RNA start site). Although this region also exhibited homologies to other transcriptional control elements, the simian virus 40 enhancer, and the adenovirus ElA enhancer, only the GC box and TATA box appear functional. These two motifs also play an essential role in vivo, although additional upstream sequences (between-139 and-55) are required for optimal transcription. DNase I footprinting, competitive gel retardation assays, and UV-photocrosslinking were used to identify Spl-like proteins of 95 and 120 kilodaltons in A92L extracts that interact with the GC box of the minute virus of mice P4 promoter.
The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).
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