DNAs, double-stranded

The double stranded DNA shown m Figure 28 14(a) contains the polynucleotide sequence (the target region) we wish to amplify The DNA is denatured by heating to... 

Artificial endonucleases, ie, molecules able to cleave double-stranded DNA at a specific sequence, have also been developed. These endonucleases can be obtained by attaching a chemically reactive group to a sequence-specific oligonucleotide. When the oligonucleotide is bound to its complementary sequence, the activation of the reactive group results in double-stranded DNA cleavage. 

Cellular protein biosynthesis involves the following steps. One strand of double-stranded DNA serves as a template strand for the synthesis of a complementary single-stranded messenger ribonucleic acid (mRNA) in a process called transcription. This mRNA in turn serves as a template to direct the synthesis of the protein in a process called translation. The codons of the mRNA are read sequentially by transfer RNA (tRNA) molecules, which bind specifically to the mRNA via triplets of nucleotides that are complementary to the particular codon, called an anticodon. Protein synthesis occurs on a ribosome, a complex consisting of more than 50 different proteins and several stmctural RNA molecules, which moves along the mRNA and mediates the binding of the tRNA molecules and the formation of the nascent peptide chain. The tRNA molecule carries an activated form of the specific amino acid to the ribosome where it is added to the end of the growing peptide chain. There is at least one tRNA for each amino acid. 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

FIGURE 4.30 Calibration curves for double-stranded DNA fragments on TSK-GEL SW and TSK-GEL PW columns. Column TSK-GEL SW or TSK-GEL PW, two 7.5 mm X 60 cm columns in series. Sample 22 fragments from Hoelll-cleaved pBR322 DNA and 6 fragments from EcoRI-cleaved pBR322 DNA. Elution 0.1 M NaCI in 0.1 M phosphate buffer, pH 7.0, plus I mM EDTA Flow rate 1.0 ml/min. Detection UV at 260 nm. 

TABLE 4.10 Recommended TSK-GEL SW and TSK-GEL PW Columns for Separating Double-Stranded DNA and RNA Fragments... 

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,... 

FIGURE 12.9 (a) Double-stranded DNA as an imaginary ladderlike structure, (b) A simple right-handed twist converts the ladder to a helix. 

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