DNA, single-stranded

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... 

The oligonucleotide population is then hybridized to single-stranded DNA and primed synthesis is carried out as described above. 

Fig. 5. Generation of mutants using single-stranded DNA. After cloning the target gene into M13, the phage is propagated in the E. coll dut, ung strain of E.

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. 

The base-specific chemical cleavage (or Maxam-Gilbert) method starts with a single-stranded DNA that is labeled at one end with radioactive (Double-stranded DNA can be used if only one strand is labeled at only one of its ends.) The DNA strand is then randomly cleaved by reactions that specifically fragment its sugar-phosphate backbone only where certain bases have been chemically removed. There is no unique reaction for each of the four bases. However,... 

If cis the concentration of single-stranded DNA at time t, then the second-order rate equation for two complementary strands coming together is given by the rate of decrease in c ... 

A graph of the fraction of single-stranded DNA reannealed c/cq) as a function of CqI on a semilogarithmic plot is referred to as a c t (pronounced cot ) curve (Figure 12.20). The rate of reassociation can be followed spectrophoto-metrically by the UV absorbance decrease as duplex DNA is formed. Note that... 

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. 

Antisense therapy means the selective, sequence-specific inhibition of gene expression by single-stranded DNA oligonucleotides. By hybridizing to the target mRNA, which results in a subsequent double-helix formation, gene expression is blocked. This process can occur at any point between the conclusion of transcription and initiation of translation or even possibly during translation. 

ASON are sequences of usually 17-30 bases of single-stranded DNA that hybridize to specific genes or their mRNA products by Watson-Crick base pairing and disrupt their function. In the case of AS-ODN (antisense oligodeoxyribonucleotides) cellular RNAseH is able to bind to the DNA-RNA duplex and hydrolyze the RNA, resulting in increased transcript turnover. Modifications to the deoxy moiety at the 2 -sugar position prohibits RNAse H action. 

A short (typically up to 80 nucleotides) single-strand DNA molecule. 

Double-stranded DNA (calf thymus) Single-stranded DNA Heat-denatured DNA Closed circular DNA Ribosomal RNA (rat liver)... 

Belotserkovskii B.P., Zarling D.A. Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes. Biochemistry 2002 41 3686-3692. 

The schematic model of the film deposited by the technique is shown in Figure 45. A single-stranded DNA layer is sandwiched between two aliphatic amine monolayers. Thus, the technique can be useful for our objectives, for it allows depisition of single-stranded DNA on practically any substrate and does not demand a large quantity of DNA, since only one monolayer will be deposited. Nevertheless, there is a question of whether DNA in such a structure will hybridize. In fact, the film contains a single-stfanded DNA monolayer between two amine monolayers, and it is questionable whether the upper amine monolayer will prevent hybridization with complementary DNA stfands. 

After hybridization and washing, the samples were stained with DAPI (4, 6-di-amidino-2-phenylindole), which apparently associates in the minor groove of double-stranded DNA (Kapuscinski 1990). DAPI from Sigma was used. Binding of DAPI to double-stranded DNA occurs with about a 20-fold fluorescence enhancement, which usually does not occur with single-stranded DNA (Haugland 1992). 

TABLE 10 Results of Fluorescent and Gravimetric Measurements of DNA Hybridization Against a Reference Containing only LB-Immobilized Single-Stranded DNA/Amine Sandwich... 

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