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Perpendicular Parallel

FIGURE 21.08 Parallel and Perpendicular DGGE

In perpendicular DGGE, the gradient of denaturing agent is at right angles to the electric field. A mixture of DNA fragments (wild-type, mutant 1 and mutant 2 in this example) is loaded into a long well that spans the entire gel. On the left where the amount of denaturant is low, none of the three DNA fragments melt and they all migrate together. In the middle of the denaturation gradient, the three DNA fragments have melted to differing extents. Mutant 1 denatures most easily and therefore migrates slower than the other fragments. Mutant 2 DNA melts the least and so migrates the farthest. On the far right of the gel, where the concentration of denaturant is greatest, all three DNA fragments are fully denatured and run together as one band of single-stranded DNA.

In parallel DGGE, the denaturing gradient runs from top to bottom, in the same direction as the electric field. Here, a mixture of mutant 1 and wild-type were loaded in the first well, and a mixture of wild-type and mutant 2 in the second. Mutant 1 denatures most easily and migrates the least. Mutant 2 is most resistant to denaturation and so migrates the fastest.

Short to medium lengths of DNA are routinely made by chemical synthesis nowadays.

Chemical synthesis of DNA is carried out with the growing chain of DNA attached to a solid support.

variety of purposes. Short stretches of single-stranded DNA are used as probes for hybridization (see below), primers in PCR (see Ch. 23) and primers for DNA sequencing (see Ch. 24). Short lengths of double-stranded DNA are made by synthesizing two complementary single-strands and allowing them to anneal. Such pieces may be used as linkers or adaptors in genetic engineering (see Ch. 22). It is also possible to synthesize whole genes, although this is more complicated (see below).

The first step in chemical synthesis of DNA is to anchor the first nucleotide to a solid support, most often a porous glass bead. Controlled pore glass (CPG) beads that have pores of uniform sizes are most commonly used. The beads are packed into a column and the reagents are poured down the column one after another. Nucleotides are added one by one and the growing strand of DNA remains attached to the glass beads until the synthesis is complete (Fig. 21.09). Chemical synthesis of DNA is performed in practice by an automated machine (Fig. 21.10). After loading the machine with chemicals, the required sequence is typed into the control panel. Gene machines take a few minutes to add each nucleotide and can make pieces of DNA 100 nucleotides or more long. Modern DNA synthesizers are also usually capable of adding fluorescent dyes, biotin or other groups used in labeling and detection of DNA (see below).

Since the DNA is made only with chemical reagents, the process requires some special modifications not necessary if biological enzymes were to manufacture the controlled pore gjass (CPG) Glass with pores of uniform sizes that is used as a solid support for chemical reactions such as artificial DNA synthesis

FIGURE 21.09 Chemical Synthesis of DNA on Glass Beads—Principle

DNA is synthesized attached to porous glass beads in a column. Chemical reagents are trickled through the column one after the other. The first nucleotide is linked to the beads and each successive nucleotide is linked to the one before. After the entire sequence has been assembled, the DNA is chemically detached from the beads and eluted from the column.

First nucleotide

Cycle 1

Cycle 1

Cycle 2

Cycle 2

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