| Hybridization-based methods
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| There are several ways in which hybridization can be used in the study of DNA, which exploit either the stringency of the hybridization of probe to target or the utility of restriction enzymes for detecting variations in nucleotide sequences.
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| OLIGONUCLEOTIDE PRIMER DESIGN |
| It is the very versatility of PCR that can be its greatest weakness. If the oligonucleotide primers are not sufficiently robust then the reliability of PCR amplification will be significantly reduced. In order to reduce the possibility of unwanted amplification, several simple rules are followed in the design of primers. Primer length should be about 20-30 nucleotides long for amplification of genomic DNA, while amplification of plasmid DNA may be performed with shorter primers. The sequence of the primer should avoid tandem repeats of nucleotides, e.g. CACACACACA, as these are common throughout the genome and will reduce the specificity of the amplification. In addition, it is important to ensure that there is no complementarity between the two primers. If the primers are complementary then they will hybridize to each other rather than the template and amplification will be inefficient. |
| By analyzing the DNA sequence of the target DNA and paying attention to these simple rules, a large percentage of the PCR amplification problems will be avoided. However, the use of computer software to perform the analysis of suitable sequences is increasingly common, and the software used is becoming extremely sophisticated and almost essential for many large-scale PCR applications. |
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