pcr假阴性和假阳性（PCR false negatives and false positives）.doc

pcr假阴性和假阳性（PCR false negatives and false positives） False negatives showed no amplification bands The key steps of PCR reaction include preparation of template nucleic acids, quality and specificity of primers, quality of enzymes and PCR cycle conditions. The reasons should also be analyzed and studied in view of the above aspects. The template template: contains protein, containing Taq enzyme inhibitor of the template, the template protein digestion without addition to the net, especially in the chromosome set of proteins in the extraction when excessive loss, or inhalation of phenol. Template nucleic acid denaturation is not complete. The enzyme and primer of good quality, no bands are likely to be digested samples, template nucleic acid extraction process is wrong, so to digestion solution effective and stable, the program should not arbitrarily change the fixed. Inactivation of enzymes: the need for new enzymes, or the simultaneous use of two new enzymes, to analyze whether or not the enzyme activity is lost or insufficient leads to false negatives. It should be noted that sometimes forget to add Taq enzyme or ethidium bromide. Primers: the quality of primers, the concentration of primers and the concentration of two primers are symmetrical, which are the common reasons for the failure or expansion of PCR bands, which are not ideal and easy to diffuse. The quality of the primers in some batches has a problem. Two primers with a high concentration and a low concentration result in asymmetric amplification with low efficiency. The strategies are as follows: 1. Choose a good primer synthesis unit. The concentration of the primer is not only to see the OD value, more attention should be paid to the original primers for agarose gel electrophoresis, must have primer bands. And two bands of the brightness should be roughly the same, such as a primer strip, a primer unhanded, then do PCR may fail, false positive The PCR amplified bands appear consistent with the target