Chapter 20 - Using in Silico Methods to Construct a Short-Tandem Repeat (STR) Deoxyribonucleic Acid (DNA) Sequence for Cloning

Abstract

Recently, the idea of forging a DNA profile in a criminal setting was explored using standard molecular biology techniques. In this experiment, modern computational tools will be used to obtain a nucleotide sequence containing a short tandem repeat (STR) of interest from a database and its complementary strand will be computed including the region upstream and downstream through the PowerPlex16 primer binding sites. Using the plasmid map for the pET-15b cloning/expression plasmid obtained from the manufacturer’s website and the DNA sequences cleaved by the restriction enzymes found in the multiple cloning site region of the plasmid, the double-stranded DNA sequence will be prepared for cloning by adding the restriction enzyme sequences to the regions upstream and downstream of the PowerPlex 16 STR primer region. The prepared DNA sequence could be purchased or produced by the polymerase chain reaction (PCR) from a template. By using the restriction enzymes to cut the DNA sequence with the primer region intact and the restriction sites added and the plasmid, the DNA can be inserted into the plasmid and ligated using DNA ligase in the lab. Extracting the DNA from the plasmid would yield an STR region amplifiable with a commercial kit.