Nucleotide sequencing polymerases

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

The gene promoter is a nucleotide sequence in DNA near the start of a gene, consisting of regulatory elements to which transcription factors and RNA polymerase bind. This leads to activation of the gene promoter and transcription of the corresponding gene. 

Promoter a specific nucleotide sequence in DNA that binds RNA polymerase and establishes the starting place for transcription. 

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

The nucleotide sequences of promoters vary considerably, affecting the binding affinity of RNA polymerases and thus the frequency of transcription initiation. Some... 

The DNA polymerases responsible for copying the DNA templates are only able to "read" the parental nucleotide sequences in the 3 —>5 direction, and they synthesize the new DNA strands in the 5 —>3 (antiparallel) direction. Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions—one in the 5 - 3 direction toward the replication fork and one in the 5 —>3 direction away from the replication fork (Figure 29.14). This feat is accomplished by a slightly different mechanism on each strand. 

In bacteria, one species of RNA polymerase synthesizes all of Ihe RNA except for the short RNA primers needed for DNA replication (RNA primers are synthesized by a specialized enzyme, primase, see p. 400). RNA polymerase is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a comple-... 

The process of RNA synthesis is called transcription. The enzyme that synthesizes RNA is RNA polymerase, which is a multisubunit enzyme. The core enzyme has four subunits—2 a, 1 p, and 1 p, and possesses 5 —>3 polymerase activity. The enzyme requires an additional subunit—sigma (a) factor—that recognizes the nucleotide sequence (promoter region) at the beginning of a length of DNA that is to be transcribed. Another protein—rho (p) factor—is required for termination of transcription of some genes. 

Initiation of transcription involves binding of the RNA polymerase to the promoter region. This sequence contains characteristic consensus nucleotide sequences that are highly conserved. These include the Pribnow box and the -35 sequence. Elongation involves RNA polymerase copying one strand of the DNA double helix, pairing C s with G s and A s (on the DNA template) with U s on the RNA transcript. Substrates are ribonucleoside triphosphates. Termination may be accomplished by the RNA polymerase alone, or may require p factor. 

Martins, A., Ribeiro, G., Marques, M. I., and Costa, J. V. (1994). Genetic identification and nucleotide sequence of the DNA polymerase gene of African swine fever virus. Nucleic Acids Res. 22, 208-213. 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

Figure 9.4. Request form for primer selection. The nucleotide sequence of a target DNA for polymerase chain reaction can be submitted for primer selection at Primer3 server.

Fig. 10.2 Schematic model for the regulation of Mn-SOD in co//.33 36) RG, regulatory gene RP, apo-repressor protein (inactive) RP-Fe3+, ferric repressor (inactive) RP-Fe2, ferrous repressor (active). Mn-SOD, nucleotide sequence of the 5 regulatory region oisodA (nucleotides —59 to +1 are shown). The +1 nucleotide designates the start point of transcription. The —35 and —10 (Pribnow box) regions for RNA polymerase binding are boxed. Fur-, and Fnr-binding sites are bracketed.

DNA is a polydeoxynucleotide and among the largest of the biological macromolecules some DNA molecules comprise more than 108 nucleotides. They contain adenine, thymine, guanine, and cytosine as the bases, and the genetic information is encoded within the nucleotide sequence, which is precisely defined over the entire length of the molecule. One of the simplest methods for determining the nucleotide sequence of DNA makes use of an enzyme, DNA polymerase, which catalyzes the synthesis of DNA. The properties of this enzyme are discussed in Chap. 16. 

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