Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Sequencing end-labeled DNA with base-specific chemical cleavages.

New M13 vectors for cloning.

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2428), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation

HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.

The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.

Mapping adenines, guanines, and pyrimidines in RNA

Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions

By exploiting the natural ability of polynucleotides to align by base pairing and using polynucleotide kinase and ligase, chemically synthesized segments have been combined into a double stranded DNA corresponding to the gene for the earliest characterized tRNA.

Total Synthesis of the Gene for an Alanine Transfer Ribonucleic Acid from Yeast

The mechanism of action of T4-induced polynucleotide kinase on short single-stranded deoxyoligonucleotides has been investigated. It was found that the phosphorylation reaction catalyzed by T, polynucleotide kinase could be reversed. Thus, a 5 ’3*P-labeled deoxyoligonucleotide and ADP on incubation with the enzyme formed [Y-~~PJATP. In addition some radioactive inorganic phosphate was produced. In the presence of ATP the major radioactive product of the reverse reaction was identified as adenosine 5’-[6-32P]tetraT he bacteriophage T.i induced polynucleotide kinase catalyzes the transfer of the y-phosphate from ATP to the 5’hydroxyl terminus of polynucleotides, oligonucleotides, 3’mononucleotides (Richardson, 1965), and N-protected deoxyoligonucleotides (van de Smde and Bilsker, 1973). The enzyme has proved to be an extremely useful tool in structural work on nucleic acids. Thus, labeling of the 5’ end groups has been used extensively for determining the end groups and terminal sequences of macromolecular DNAs (Weiss and Richardson, 1967 ; Jacquemin-Sablon and Richardson, 1970; Wu and Kaiser, 1967) and for structural study of thc cohesive ends of the bacteriophage X D N A (Wu and Kaiser, 1968; Richardson et al., 1908). Similarly, the fingerprinting of short oligonucleotides (pyrimidine tracts from DNAs, nuclease digests of tRNAs) is facilitated by the labeling of the 5’-end groups (SzCkely and Sanger, 1969: Southern, 1970: Murray, 1973; Simsek er al., 1973). Further, the techniques of introducing label at the termini of polynucleotide chains facilitate studies of the polynucleotide ligase and of D N A enzymology (Weiss et al., 1968b; Gefter et cd.: 1967; Olivera and Lehman, 1967) and 5’-phosphomonoesterases (Becker and Hurwitz, 1967 ; Weiss et al.?

Physical characterization and simultaneous purification of bacteriophage T4 induced polynucleotide kinase, polynucleotide ligase, and deoxyribonucleic acid polymerase.

A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

https://science.sciencemag.org/content/sci/241/4865/551.full.pdf

J.H. van de Sande

Papers

Total Synthesis of the Gene for an Alanine Transfer Ribonucleic Acid from Yeast

Physical characterization and simultaneous purification of bacteriophage T4 induced polynucleotide kinase, polynucleotide ligase, and deoxyribonucleic acid polymerase.

Parallel stranded DNA

Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast.

Reversal of bacteriophage T4 induced polynucleotide kinase action.