PCR Template DNAThe DNA in a PCR reaction comprises two types:
- the target sequence to be amplified
- the non-target DNA (also called the "burden" DNA
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The application of molecular technology in clinical diagnosis in two key diagnostic areas: cancer and infectious diseases.
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Provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and supplies detailed technical insights into the underlying principles, methods and practice of real-time PCR.
Problems also occur when the ratio of the target DNA to the burden DNA is very low, for example the amplification of a 500 bp fragment from the human genome (1 to 6 x 106). A better ratio is between 1:1 and 1:1 x 104. A ratio of 1:1 is achieved in a reamplification reaction and a ratio of about 104 is achieved when amplifying from the Escherichia coli genome.
When the total amount of the DNA in a PCR reaction is extremely small, there is an increased likelihood of its loss owing to any conceivable cause (clotting, adsorption, chemical or enzymatic degradation). Furthermore, a small amount of target DNA leads to an increased risk from contaminating DNA from impurities on anything that can come into contact with the DNA solution. In this respect, both the DNA diluent, the dust floating in the air, exhalations and even particles of skin or hair from your body should not be disregarded, as these can carry both the DNA and the DNA-degrading substances. Nucleases are probably as the major cause of DNA degradation in a PCR procedure. They are abundant on the surface of the human skin and can be present everywhere else too. Mild autoclaving of the DNA diluent and everything that comes in regular, occasional, or accidental contact with buffers and solutions will destroy both the nucleases and comtaminating DNA. If you suspect problems of this nature, wear gloves, a surgeon's cap, and a face mask. Also, wash the working space with an oxidizing substance such as (6% H202).
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