DNA-Sequencing

Various experimental conditions have been optimized for DNA sequencing on a glass chip. These conditions include separation matrix (denaturing 3-4% LPA), separation temperature (35—40°C), channel length (7.0 cm), channel depth (50 pm), injector parameters (100-pm or 250-p.m double-T injector, 60-s loading time). These optimal conditions facilitated the one-color detection of separation of 500 bases of M13mpl8 ssDNA in 9.2 min, and four-color detection of 500-base separation in 20 min [548]. 

Further optimization of DNA sequencing of M13mp 18 DNA (up to 550 bases) on a microfabricated glass chip was achieved on separation buffer composition (no borate), sieving polymer concentration ( 2% LPA), device temperature (  

When a longer CE channel (40 cm) on a glass chip was used, the average DNA read length (four-color-detection) was successfully increased to 800 bases in 80 min (98% accuracy) for M13mpl8 DNA (or the DNA with a 2-kbp human chromosome 17 insert). For comparison, a commercial capillary-based DNA sequencer took 10 h to complete the same 800-base analysis [968]. Moreover, DNA sequencing up to 700 bp (with 98% accuracy) has been achieved in 40 min using an 18-cm plastic microchannel [969]. 

A hybrid device was constructed for DNA sequencing. Other than a micro-channel, a capillary tube was interfaced to a glass chip for separation [114]. 

In a later report, a 96-lane CE glass chip was used to carry out DNA sequencing. Tapered turns were employed to increase the channel length, and hence the DNA read length [972], More recently, a 384-lane plate for DNA sequencing has also been reported [973], 

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ITS Sequence tagged site. A short DNA sequence that occurs just once in the human genome and whose location and base sequence are known... 

PCR can also be used to modify DNA sequences using primers differing at one or several positions from the target sequence. This is possible because PCR does not require perfect complementarity of a primer to the sequence flanking the target. Since all of the PCR products contain the primer sequence, an insertion or deletion can thus be incorporated into the product by modifying a primer. It is also possible to add new sequences to the 5 -ends of the primers. Modified or additional genetic information may thus be multiplied and transr ported. 

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25-1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. 

DNA sequence data have been used to investigate inherited diseases such as hemophilia and muscular dystrophy, and also in cancer research. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

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. 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

DNA sequence information is the starting poiat for other appHcations, including the expression of a gene product, the search for related sequences ia biological samples, in vitro mutageaesis of the sequeace, and stmcture—function studies of gene expression. 

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

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