Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Chemical Synthesis, Nucleotide Sequencing, and DNA Amplification
Conclusion

Along with Gene cloning, Methods such as Chemical DNA Synthesis, DNA Sequencing, and the Polymerase Chain Reaction (PCR) rank among the most crucial techniques in molecular biotechnology.

The purpose of chemical synthesis is to produce single-stranded DNA molecules in vitro. Success here relies entirely on achieving a high efficiency of phosphodiester bond formation; otherwise, the process will yield only a very small quantity of molecules of the desired size. Molecules synthesized in vitro are typically 20—30 NUCLEOTIDES long and rarely exceed 100 nucleotides. To obtain double-stranded molecules, the complementary strands are synthesized separately and then annealed. DNA produced in this manner is used as probes for screening Genomic Libraries; as linkers and adaptors in gene cloning; for in vitro mutagenesis; and for constructing genes for subsequent cloning.

Solving biotechnological and other related problems very often requires knowing the complete nucleotide sequence of a cloned gene. Several methods are available for sequencing; one of them is the dideoxy method developed by Sanger and coworkers. It is based on terminating chain synthesis once a dideoxynucleotide has been incorporated. Because such a nucleotide lacks a 3'-hydroxyl group, further chain elongation becomes impossible. For sequencing, four separate DNA synthesis reactions are carried out simultaneously in different tubes, each containing one of the four dideoxynucleotides. The reaction products are then separated by gel Electrophoresis, subjected to autoradiography, and The nucleotide sequence of the synthesized DNA fragment is "read" from the autoradiogram.

Sequencing can also be performed using an M13 phage-based system. A DNA fragment of up to 500 nucleotides to be sequenced is inserted into the phage DNA. This recombinant DNA is easily obtained in single-stranded form and used as a template for sequencing the insert. Alternatively, double-stranded Plasmids containing the cloned DNA can also be used.

To determine the nucleotide sequence of extended cloned segments, researchers first select a synthetic oligonucleotide primer complementary to the region adjacent to the insert and sequence the first 250—300 nucleotides using the dideoxy method. Based on these sequencing results, a second primer is synthesized to determine The sequence of the next 250—350 nucleotides of the cloned segment, and so on. This method, known as "primer-walking" (or "primer-directed sequencing"), allows the sequencing of long DNA fragments without The Need for subcloning, as is required in the M13 phage system.

The PCR method revolutionized biotechnology by making it possible to amplify desired DNA segments in vitro millions of times over. The Procedure is as follows: two primers are chosen that hybridize to the DNA regions flanking the target sequence. The DNA is denatured, the single-stranded molecules are annealed with an excess of added primers, and DNA synthesis is carried out in vitro. To facilitate the synthesis, a thermostable DNA polymerase is used that is not destroyed at Denaturation temperatures (95 °C). Denaturation, primer annealing, and synthesis are then repeated cyclically for about 30 rounds. By this stage, the reaction mixture is dominated by fragments having one primer sequence at one end and a sequence complementary to the second primer at the other. PCR can be used to detect pathogenic microorganisms in various biological samples; to obtain large quantities of specific DNA fragments for cloning; to amplify the 5'- and 3'-ends of specific mRNAs; to synthesize genes; and to identify deletions or insertions in genes responsible for specific Genetic Disorders.

References

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Fox D. К., В. Westfall, М. Nathan, A. J. Hughes, Jr., A. Rashtchian, D. M. Schuster. 1996. Striding new distances with 5'RACE: long 5'RACE of human APC and TSC-2 cDNA. Focus 18: 33—37.

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Mullis К. B., F. Ferre, R. A. Gihbs (ed.). 1994. The Polymerase Chain Reaction. Birkhäuser, Boston, Mass.

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1988. Primer-directed enzymatic Amplification of DNA with a thermostable DNA polymerase. Science 239: 487—491.

Sanger F., S. Nicklen, A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sei. USA 74: 5463—5467. Schuster D. M., G. W. Buchman, A. Rashtchian.

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Review Questions

1. Assuming your new DNA synthesizer has an average nucleotide coupling efficiency of 98.5%, what will the yield of the product be

if you synthesize a Hybridization probe that is 50 nucleotides long?

2. What two strategies for the chemical synthesis of a 0.5-kb gene can you propose? Which one would you prefer?

3. WHAT IS A linker? Where is it used?

4. What are dideoxynucleotides? How are they used to determine the nucleotide sequence of DNA?

5. Why can the nucleotide sequence be determined only for single-stranded DNA?

6. How is the nucleotide sequence of cloned DNA determined using an M13 phage-based vector system?

7. A single Hair of a suspected criminal is found at a crime scene. It contains 10–20 picograms (10^-12 g) of DNA. To characterize such a small amount of DNA and determine whether its nucleotide sequence is identical to that of the suspect's DNA, 10–100 nanograms (10^-9 g) of DNA are required. How can it be obtained? What information do you need to gather before taking any action?

8. What are a "long template" and a "short template", and how does the ratio between them change as the number of PCR rounds increases?

9. How are genes synthesized using PCR?

10. How can the ends of an mRNA molecule be "converted" into cDNA?