Robert C.A. Yang

[10] Elution of DNA from agarose gels after electrophoresis

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.

The nucleotide sequence of a new human repetitive DNA consists of eight tandem repeats of 66 base pairs

Three cloned human DNA fragments obtained from a fibroblast genomic DNA were sequenced and identified as containing members of the well-known 300-bp Alu family of interspersed, middle-repetitive DNA sequences. One of these cloned DNA fragments, p16, also contains members of a new repetitive DNA family, which repeats several thousand times in the human genome. Each member of the new 528-bp family consists of eight tandem repeats of a 66-bp sequence. An AluI recognition site is present at the same location in each repeat, and a 25-bp sequence occurs twice (as a tandem repeat) in each of the eight repeats. There is no sequence homology between the new 528-bp family and the 300-bp Alu family, and the new family lacks the flanking 7- to 20-bp direct repeats as well as the dAMP-rich sequences characteristic of the 300-bp Alu family. Construction of a putative evolutionary tree indicates that six duplication events are needed to give rise to the eight tandemly repeated 66-bp units in the new 528-bp family.

BK virus DNA sequence coding for the amino-terminus of the T-antigen

Abstract Nucleotide sequence between map positions 0.540 and 0.604 on the human papovavirus BK(MM strain) genome has been determined. Following a potential initiation ATG signal at 0.595 map position, a unique reading frame is identified which can code for at least 95 amino acids. The first 10 amino acids deduced therefrom at the amino-terminus coincides with those of the T-antigen molecules of simian virus 40. Between amino acids 11–74, there is a 78% homology of nucleotide sequence and an 84% homology of amino acid sequence. An unusual nucleotide sequence with a two-fold rotational symmetry preceding the aforementioned ATG signal has been located in BKV(MM) DNA as well as in SV40 DNA, and a secondary structure with a possible regulatory function is proposed.