Enzyme-catalyzed polymerization of nucleotides is a key step in DNA identification. The microfluidic device shown in Figure SN7.1 is used to identify DNA strands. It was developed by Professor Mark Burns's group at the University of Michigan.
Figure SN7.1 Microfluidio device to identify DNA. Courtesy of Science, 282 p. 484 (1998).
In order to identify the DNA, it's concentration must be raised to a level it can be quantified. This increase is typically accomplished by replicating the DNA in the following manner. After a biological sample (e.g., purified saliva, blood) is injected into the micro device, it is heated and the hydrogen bonds connecting the DNA strands are broken. After breaking, a primer attaches to the DNA to form a DNA primer complex, DNA*. An enzyme E then attaches to this pair forming the DNA* enzyme complex, DNA*•E. Once this complex is formed, a polymerization reaction occurs as nucleotides (dNTPs--dATP, dGTP, dCTP, and dTTP--N) attach to the primer one molecule at a time as shown in Figure SN7.2. The enzyme interacts with the DNA strand to add the proper nucleotide in the proper order. The addition continues at the enzyme moves down the strand attaching the nucleotides until the other end of the DNA strand is reached. At this point the enzyme drops off the strand and a duplicate, double-stranded DNA molecule is formed. The reaction sequence is
Figure SN7.2 Replication Sequence.
The schematic in Figure SN7.2 can be written in terms of single step reactions where N is one of the four nucleotides.
Complex Formation:
DNA + Primer ® DNA*
Nucleotide addition/polymerization
The process then continues much like a zipper as the enzyme moves along the strand to add more nucleotides to extend the primer. The addition of the last nucleotide is
Where i is the number of nucleotide molecules on the original DNA minus the nucleotides in the primer. Once a complete double-stranded DNA is formed, the olymerization stops, the enzyme drops off, and separation occurs.
Here 2DNA strands really represent one DNA two-strand helix. Once replicated in the device, the length of the DNA molecules can be analyzed by electrophoresis to indicate relevant genetic information.
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