Question

An unknown DNA molecule was cleaved using several restriction enzymes individually and in various combinations. The DNA fragment sizes were determined by agarose gel electrophoresis and the restriction enzyme recognition sites were mapped. Subsequently, the DNA was sequenced, and an extra recognition site was found for one of the enzymes. However, all the other mapping data was consistent, within experimental errors, with sequence data. What are the simplest explanations for this discrepancy?

Answer 1

Answer:

DNA digestion was not complete.

Explanation:

When a particular DNA needs to be used for the experiment, it is firstly processed by restriction digest. A restriction digest is a procedure for DNA preparation for further examination and manipulation. It is defined as a technique used for separating DNA fragments. This separation is being done using specific enzymes that will bond to specific sites and locations to the DNA so all DNA fragments will be a similar size. These enzymes are called restriction endonucleases or restriction enzymes and are extracted from bacteria for this particular cause. Once fractured or digested, DNA fragments may be amplified by PCR (polymerase chain reaction) and used for further experimentation.

When DNA is sequenced, all recognition sites (on enzymes) are visible and detectable. However, if an extra recognition site is found after sequencing, it means that initial mapping was not completed (assuming the DNA sequence has no errors). Initial restriction digestion was not completely done, so DNA is stuck in the middle of the process, in various degrees of supercoiling and digestion.