We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

Rapid and simple method for purification of nucleic acids.

A method for obtaining sequence information directly from plasmid DNA is presented. The procedure involves the rapid preparation of clean supercoiled plasmid DNA from small bacterial cultures, its complete denaturation by alkali, and sequence determination using oligodeoxyribonucleotide-primed enzymatic DNA synthesis in the presence of dideoxynucleoside triphosphates. The advantages of the method include speed, simplicity, avoidance of additional cloning steps into single-stranded phage M13 vectors, and hence applicability to sequencing large numbers of samples.

Supercoil sequencing: a fast and simple method for sequencing plasmid DNA

Dideoxy sequencing method using denatured plasmid templates.

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: Application to sequencing the corn mitochondrial 18 S rDNA

https://www.cell.com/cell/pdf/0092-8674(83)90133-2.pdf

Structures of P transposable elements and their sites of insertion and excision in the Drosophila melanogaster genome

Publisher Summary This chapter discusses the elution of deoxyribonucleic acid (DNA) from agarose gels after electrophoresis. The studies of genome structure and function rely heavily on the isolation and analysis of the defined DNA fragments. Gel electrophoresis is a simple, high-resolution method of separating specific DNA fragments on the basis of size. Agarose gels at concentrations of 0.1–2.5% resolve DNA from 150–880,000 base pairs, whereas acrylamide gels, ranging from 3–20%, afford a good resolution of fragments in the size range of 10–2,000 base pairs. The chapter describes three new or revised methods for the recovery of DNA from agarose gels. The evaluation is based on the simplicity, speed, yield, and amenability of the purified DNA. The first method involves the electroelution of DNA into slots. The second method involves the electroelution of DNA onto dialysis membranes. The third method describes the process of dissolving gel slices in perchlorate solution.

Elution of DNA from agarose gels after electrophoresis.

The complete DNA sequence of the human papovavirus BK is presented. From the 4963 base-pair sequence of BK virus (MM strain), the amino acid sequence of at least five proteins can be deduced: a T antigen and a t antigen, which share amino terminal peptides; proteins VP2 and VP3, which share 232 amino acids; and protein VP1, whose coding sequence overlaps those for VP2 and VP3 by 113 nucleotides but is read in a different frame. The gene loci and the arrangement of genes are strikingly similar in BK virus and simian virus 40 (SV40). The sequence of the deduced proteins in BK virus shares 73 percent amino acid homology with those in SV40, whereas the DNA sequence of the two viruses shares 70 percent homology, suggesting close evolutionary relationship. However, the repeated DNA sequences in the noncoding regions of these viruses are different.

https://science.sciencemag.org/content/sci/206/4417/456.full.pdf

BK virus DNA: complete nucleotide sequence of a human tumor virus

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.

An Improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid

Extensive physical mapping revealed that approximately 90% of the genomes of BKV(prototype, WT) and BKV (MM strain) are identical or closely related. Nucleotide sequences of the non-homologous regions and a large portion of the homologous regions have been determined for both genomes. The coding sequence of small t antigen of BKV(MM) is 216 nucleotides shorter than that of BKV(WT), even though no differences in biological function of the t antigen was observed. Both genomes contain three similar sets of 44-61 base-pair repeated sequences. However, the DNA sequence of the tandem repeats is totally different between BKV (human cell as host) and SV40 (monkey cell as host). On the other hand, the region between the N-terminus of the T antigen genes and the origin of replication is dominated by a similar set of palindromic sequences in BKV and SV40 DNA. There is also extensive homology between the regions which code for proteins in BKV and SV40, suggesting a close evolutionary relationship.

Comparative study of papovavirus DNA: BKV(MM), BKV(WT) and SV40

The primary nucleotide sequence of three regions of BK virus (BKV) variant (MM) DNA has been determined. The region between map positions 0.715 and 0.900 includes the initiation points and partial coding sequences of the putative VP2 and VP3 proteins of BKV(MM), the amino acid sequences of which show over 80% homology with those of VP2 and VP3 of simian virus 40. The sequence of a potential leader protein X, 66 amino acids long for BKV(MM) and 62 long for simian virus 40, is also deduced. The regions between 0.595 and 0.398 and 0.310 and 0.175 include the coding sequence for the entire small t antigen and most of the large T antigen of BKV(MM). The DNA sequence within these regions comprises over 50% of the complete BKV(MM) genome and shows a 70% sequence homology with the corresponding regions of simian virus 40 DNA. This high degree of homology is at variance with the reported homology values of 11--20% estimated by hybridization measurements of heteroduplex analyses. Possible explanations for the discrepancy are discussed.

Robert C. A. Yang

Papers

Elution of DNA from agarose gels after electrophoresis.

BK virus DNA: complete nucleotide sequence of a human tumor virus

An Improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid

Comparative study of papovavirus DNA: BKV(MM), BKV(WT) and SV40

BK virus DNA sequence: extent of homology with simian virus 40 DNA.