Sequencing DNA fragments

Figure 6.4. Strategy of the Chain-Termination Method for Sequencing DNA. Fragments are produced by adding the 2, 3 -dideoxy analog of a dNTP to each of four polymerization mixtures. For example, the addition of the dideoxy analog of dATP (shown in red) results in fragments ending in A. The dideoxy analog cannot be extended.

Figure 6.17. M13 Phage DNA, a Cloning and Sequencing Vector. M13 phage DNA is very useful in sequencing DNA fragments by the dideoxy method. A double-stranded DNA fragment is inserted into Ml 3 RF DNA. Synthesis of new strand is primed by an oligonucleotide that is complementary to a sequence near the inserted DNA.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND ELECTROPHORESIS 13.2.5 Sequencing DNA Fragments... 

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

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 lac repressor monomer, a chain of 360 amino acids, associates into a functionally active homotetramer. It is the classic member of a large family of bacterial repressors with homologous amino acid sequences. PurR, which functions as the master regulator of purine biosynthesis, is another member of this family. In contrast to the lac repressor, the functional state of PurR is a dimer. The crystal structures of these two members of the Lac I family, in their complexes with DNA fragments, are known. The structure of the tetrameric lac repressor-DNA complex was determined by the group of Mitchell Lewis, University of Pennsylvania, Philadelphia, and the dimeric PurR-DNA complex by the group of Richard Brennan, Oregon Health Sciences University, Portland. 

Figure 9.3 Comparison of the consensus nucleotide sequence of the TATA box (a) and the sequences of the DNA fragments used in the crystal structure determinations of the TATA box-binding proteins from yeast (b) and the plant Arabidopsis thaliana (c).

Figure 9.19 Nucleotide sequence of the 21-base pair DNA fragment cocrystalUzed with the DNA-binding domain of p53. The p53 binds in a sequence-specific manner to the shaded region.

Interactions that are not sequence specific are also an Important part of the binding and occur between the sugar and phosphate residues of the DNA and the side-chain and main-chain atoms of the protein. In the crystals the DNA fragment retains the B-DNA structure with only minor distortions. 

Figure 10.2 (a) Amino acid sequence of a fragment of the Zif 268 protein that contains three zinc fingers. Residues forming the p strands and a helices are red and green, respectively, and those involved in the turn between the last p strand and the a helix are blue, (b) The nucleotide sequence of the DNA fragment that was used in the x-ray structure determination of the Zif 268 fragment complexed with DNA. 

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