Sequencing single-stranded

Chapter 4 describes the application of primed synthesis methods to sequencing single stranded DNAs prepared by cloning restriction fragments into derivatives of the single stranded DNA phage... 

A method for sequencing single stranded cloned DNA in both directions by the dideoxynucleotide-chain termination procedure (Hong, G.F., Bioscience Reports 1, 243-252 (1981)... 

Nakagami et al. (1991) proposed a different universal probe system. In their procedure, phagemids are used to obtain strand-specific ss DNA molecules, one with the insert complementary to the target sequence and a second without the insert and with the opposite polarity (Fig. 7.23). The ss DNA without insert is labeled with any label (usually a secondary label) and this labeled DNA is hybridized with the complementary strand containing the insert. This leaves only the probe sequence ss whereas the tag sequence is ds. This method has several advantages (i) like the sandwich method it leaves the probe sequence single-stranded (ii) hybridization of the tag sequences in solution instead of on the solid phase as in a sandwich assay is much faster (iii) no fragmentation is required. 

Figure 8.4.3 Sequence- and strain-specific transcription of DNA into an mRNA sequences. Single-strand sense mRNA is formed. Antisense mRNA could form a double strand with such a molecule.

Section 28 14 The nucleotide sequence of DNA can be determined by a technique m which a short section of single stranded DNA is allowed to produce its complement m the presence of dideoxy analogs of ATP TTP GTP and CTP DNA formation terminates when a dideoxy analog is incorporated into the growing polynucleotide chain A mixture of polynucleotides dif fermg from one another by an incremental nucleoside is produced and analyzed by electrophoresis From the observed sequence of the comple mentary chain the sequence of the original DNA is deduced... 

Pig. 7. The PGR reaction, showiag amplification of a sequence located between two primers where ( ) represents a primer, (—) a single strand of DNA,... 

Fig. 9. Mutagenesis by a synthetic oligonucleotide of a cloned sequence available in single-stranded form (a) single-stranded M13 template containing uracil (U) residues (b) double-stranded product, uracil residues are not mutagenic (c) strong selection for M13 phages containing mutation of interest (23).

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Moreover, disposable electrochemical sensors for the detection of a specific sequence of DNA were realised by immobilising synthetic single-stranded oligonucleotides onto a graphite or a gold screen-printed electrode. Tire probes became hybridised with different concentrations of complementary sequences present in the sample. 

The sequence of bases in DNA is recorded as a sequence of complementary bases in a single-stranded mRNA molecule. 

If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other. The degree of hybridization is a measure of the sequence similarity or relatedness between the two species. Depending on the conditions of the experiment, about 25% of the DNA from a human forms hybrids with mouse DNA, implying that some of the nucleotide sequences (genes) in humans are very similar to those in mice. Mixed RNA DNA hybrids can be created in vitro if single-stranded DNA is allowed to anneal with RNA copies of itself, such as those formed when genes are transcribed into mRNA molecules. 

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