| It is sometimes necessary to quantify amounts of RNA or DNA in a clinical sample. For example, in cases of human immunodeficiency virus (HIV) infection, knowledge of the amount of viral RNA in the bloodstream can be a valuable guide to prognosis and treatment. In standard PCR reactions, one usually examines the DNA produced after 30 or more cycles, when it has been amplified 1 000 000 000 times or more. This is a useful qualitative test, but is not good for quantification. For example, the amount of PCR products approximately doubles each cycle so small differences in efficiency at earlier cycles result in large differences at later cycles. In addition, all reactions eventually reach a plateau phase due to many reasons, including the accumulation of end-products that inhibit the reaction, and many reactions will have reached plateau phase before 30 cycles. To circumvent many of these problems, real-time PCR machines have been developed which measure how much product is formed at very early stages in the reaction when it is proceeding at the maximal rate. If there is more target DNA in a particular sample, fewer cycles will be needed to make a defined amount of product, so the number of cycles needed to make a defined amount of product is a measure of how much of that DNA sequence was present at the beginning of the PCR. In real-time PCR, the formation of product is followed either by using a dye which fluoresces brightly when bound to double-stranded DNA, or by using some form of fluorescently-labeled hybridization probe. The amount of product is measured continuously at the end of every PCR cycle, e.g. by measuring fluorescence, using a probe that intercalates into double-stranded DNA and changes or increases its fluorescence on binding. Sensitive optics and powerful electronics are used so that very small amounts of product can be detected at early cycles and the process can easily be automated. This is a convenient way to quantify accurately, sensitively and quickly small amounts of DNA or RNA (which must first be transcribed to cDNA) and the technology is rapidly moving from research laboratories to clinical applications. |
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| There are a myriad of DNA techniques available for the analysis of every aspect of DNA metabolism and function. However, regardless of the complexity of the methods, they all rely on the basic principles of hybridization and polymerization by appropriate enzymes. There is no doubt that the most significant recent advance in the study of DNA has been the discovery and automation of PCR. PCR
is now used widely in all aspects of biomedical research, in particular, the study of human genetics. Whether in the diagnostic laboratory, or in the search for new disease genes, analysis has been totally transformed by PCR. There are now many hundreds of human genetic diseases whose diagnosis can be made by a single PCR-based method on DNA from a single blood sample. In many cases the PCR-based diagnosis has replaced complex biochemical tests or allowed a definitive diagnostic test to be available for the first time, e.g. in Huntington's disease. As a result of the expansion of the use of PCR and the
versatility of PCR-based techniques, the use of Southern blotting and RFLPs has decreased. However, the basic principles of Southern blotting are at the center of all protocols that require radioactive labels to identify the reaction products, and an understanding of the principle of probe-target hybridization is essential.
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| The virtual completion of the Human Genome Project, within just 50 years of the discovery of the three dimensional structure of DNA, stands as one of mankind's great achievements. Of course this vastly simplifies the study of both normal and mutant genes, since the information to design PCR primers and to predict restriction enzyme cut-sites is now available for all genes.
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| Recombinant DNA technology has replaced complex biochemical assays as the major biological scientific technology in most laboratories. The subject is continually evolving, but at its center are several key principles that are modified to fit the application of a particular method. Understanding these key steps is the key to grasping the seemingly abstract nature of some of the newer techniques.
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- A 14-year-old female patient appeared to be undergoing normal sexual development, except for rather scant body hair. Her complaint was that she has never had a menstrual period. Physical examination indicated a very short vagina ending in a blind pouch. How could you use the human genome sequence to design primer pairs that would amplify exons of the androgen receptor to check for possible mutations that could account for androgen insensitivity syndrome?
- New human pathogenic viruses appear with some regularity. If you were in charge of a large research group, how could you apply cell-based cloning techniques to establish the sequence of a new viral DNA and then suggest possible viral proteins that could be expressed to use in a recombinant vaccine?
- An unsorted collection of clones derived from the DNA of an organism is called a genomic library. Similarly, an unsorted collection of clones derived from the mRNA of a particular tissue is termed a cDNA library. Why are cDNA libraries most useful if the goal is to express a specific human pituitary protein in yeast cells, but a genomic library would be required if the aim was to characterize binding sites for transcription factors in the promoter of the same gene?
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Braude P, Pickering S, Flinter F, Ogilvie CM. Preimplantation genetic diagnosis. Nat Rev Genet 2002;3:941-953.
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| Chance RE, Frank BH. Research, development, production, and safety of biosynthetic human insulin. Diabetes Care 1993;16 Suppl 3:133-142.
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| Cockerill FR. Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Arch Pathol Lab Med 2003;127:1112-1120.
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| Copland JA, Davies PJ, Shipley GL, Wood CG, Luxon BA, Urban RJ. The use of DNA microarrays to assess clinical samples: the transition from bedside to bench to bedside. Recent Prog Horm Res 2003;58:25-53.
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| Guo, OM. DNA microarray and cancer. Curr Opin Oncol 2003;5:36-43.
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| Ivnitski D, O'Neil DJ, Gattuso A, Schlicht R, Calidonna M, Fisher R. Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents. Biotechniques 2003;35:862-869.
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| Primrose SB, Twyman, R. Principles of Genome Analysis and Genomics. Oxford: Blackwell, 2002.
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| Tuzmen S, Schlechter AN. Genetic diseases of hemoglobin: diagnostic methods for elucidating beta-thalassemia mutations. Blood Rev 2001;15:19-29.
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| http://www.ncbi.nlm.nih.gov National Center for Biotechnology Information: an entry site that leads to full genome sequences for human and mouse as well as much other sequence-related information.
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