The Sequencing of DNA

Gene, the sequence of DNA that codes for a complete protein. 

Manual Automatic Techniques Are Available to Determine the Sequence of DNA... 

So, to summarize, the hole transfer rate between Py and Ptz was determined using the transient absorption measurements of Py + and Ptz +, and we have shown that the hole transfer rate from Py + to Ptz depends on the distance and sequence between Ptz and Py. In other words, the hole transfer rate in DNA is modulated by designing the sequence of DNA and conjugated molecules. It is suggested that DNA may be utilized as a molecular wire by introducing several organic molecules at the appropriate site in DNA. 

The sequence of DNA recognized by a specific restriction endonuclease is often palindromic. A palindrome is something that reads the same way backward and forward (Fig. 6-2). The sequence of the bottom strand read in the 5 to 3 direction is the same as that of the top strand read in the 5 to 3 direction. The usual analogy for a verbal palindrome is a sentence that reads the same way backward and forward. Madam, I m Adam is the usual example. It s not exactly the same way for DNA palindromes. The top strand does not read the same from the left as from the right the top strand read from left to right is the same as the bottom strand read from right to left. 

Pieces of DNA, for example genes or cDNA probes, are multiplied using a method based on in vivo replication in bacterial cells. The sequence of DNA is incorporated into a vehicle or vector which transports it into a cell host, usually E. coli. As the bacteria culture grows, the vector also replicates so producing more of the required sequence of DNA. Two of the naturally occurring types of DNA molecule which can be used as vectors are plasmids and viral chromosomes. 

Geneticists use maps to describe the location of a particular gene on a chromosome. One type of map uses the cytogenetic location to describe a gene s position. The cytogenetic location is based on a distinctive pattern of bands created when chromosomes are stained with certain chemicals. Another type of map uses the molecular location, a precise description of a gene s position on a chromosome. The molecular location is based on the sequence of DNA building blocks (base pairs) that make up the chromosome. 

Epigenetic is a term used to describe a state of gene expression that is mitotically and meiotically inherited without any change in the sequence of DNA. Epigenetic mechanisms are mainly of two classes (1) the DNA may be modified by the covalent attachment of a moiety that is then perpetuated. (2) a self-perpetuating protein state may be established (Zelent et al, 2004). The two most studied epigenetic phenomena are DNA methylation and histone tail modifications (Mai et ai, 2005). 

Because the sequencing of DNA has become so straightforward, genes of several ribosomal proteins have been sequenced to allow an independent determination of the primary sequence of some of the proteins (Post et ai, 1979 Olins and Nomura, 1981). These studies have confirmed the sequences previously elucidated by protein chemical techniques. In the case of SI (the largest of all E. coli proteins), the combination of amino acid- and nucleotide-sequence determinations was used to provide the sequence (Schnier et ai, 1982). 

Mutations change the sequence of DNA bases and may thus lead to cellular dysfunction or disease. 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

The collecting, organizing, and indexing of sequence information into a database provides the scientist with a wealth of information on human genome and proteome. What makes this database so useful and powerful is its analysis, which may lead to information indicating that the sequence of DNA in question does not always constitute only one gene it may contain several genes. 

To avoid too fast an energy transfer and enhance the selectivity of the laser excitation of the chosen sites in large biomolecules, it seems very promising to use their chemical labeling. This is especially important in the mapping of the sequences of DNA nucleotide bases having very close spectral properties. The first experiments on the MPI of dye-labeled nucleic acid bases were quite a success [11]. 

The specific properties of any protein are due to the specific sequence of amino acids in its polypeptide chains. This sequence is determined by the genetic information carried by the sequence of DNA nucleotides. DNA transfers the information to messenger RNA, which serves as the template for protein synthesis. 

Figure 9.9. Sequence alignment with BioEdit. The sequences of DNA encoding preprosomatostatin mRNA are aligned to identify the consensus sequence.

The chapter begins with a discussion of the structure of nucleosides and nucleotides. Then the structure of the nucleic acids, DNA and RNA. the polymers formed from nucleotide monomers, is presented. The function of these polymers in the replication, transcription, and translation of genetic information is briefly addressed. Next, the organic chemistry involved in determining the sequence of DNA is presented. Finally, the synthesis of small DNA molecules in the laboratory is discussed. 

Understand the chain-terminator method for determining the sequence of DNA. 

The sequence of DNA bases is of great importance in heredity. In fact, the sequence of bases in one strand has a complementary relationship to the sequence of bases in the other strand. In other terms, information contained in the sequence of one strand is conserved in the second strand. 

DNA probes (Section 19.18) whose sequences are complementary to the mutated sequences are used to examine an individual s genome, or to make comparisons of the sequence of DNA bases in one of their genes with a normal version of that gene. There are currently more than 1000 genetic tests, with an increasing number becoming available commercially. Some examples include ... 

The above task, once accomplished, allows the researcher to view the sequence of DNA bases (read from the polyacrylamide gel) in terms of a series of consecutive codons (triplets of bases that code for amino acids). The next task is to search for the translation start site and the translation stop site. Once the entire sequence of the polypeptide has been acquired from the DNA sequence, the researcher may compare the amino acid sequence with those of all known proteins. This task, which is performed on the computer, can reveal whether or not the polypeptide being studied is closely related to another, better characterized polypeptide. If a close match is found, the researcher may gain insight into the functions of the gene being sequenced. 

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