There are several techniques for further analyzing amplified DNA. One of the simplest approaches involves detecting changes in DNA cutting patterns following digestion with a restriction enzyme, known as restriction fragment length polymorphism (RFLP). Restriction enzymes recognize specific base sequences with high precision and cut the DNA at these exact sites. Consequently, base substitutions may lead to the gain or loss of a restriction site, resulting in DNA fragments of varying lengths compared to the wild-type (normal) sequence after digestion. These fragments can be visualized by separating them based on size in an agarose gel and staining with a fluorescent dye. The RFLP technique is commonly used to identify known base substitutions and to verify newly discovered variants.
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