Dna Polymerase

New DNA

Elongation

FIGURE 23.26 Rolling Circle Amplification of DNA—Linear Version

Just like rolling circle replication in bacteria, RCAT produces many copies of a circular target DNA. This process only requires one primer for DNA polymerase, and the temperature does not need to be elevated. The result is a long linear piece of DNA.

Amplification of DNA by the rolling circle mechanism avoids the high temperature step of PCR.

the most promising is Rolling Circle Amplification Technology (RCAT) (marketed by Molecular Staging, New Haven, CT).

This technique is based on the rolling circle mechanism for DNA replication used by many plasmids and viruses (see Ch. 17); therefore, a circular DNA template is needed. This process occurs at a single temperature and does not require thermostable DNA polymerase. In linear RCAT, a DNA primer binds to the circular DNA, which is then copied many times to give a long single-stranded product that may consist of up to 100,000 tandem repeats of the target sequence (Fig. 23.26). Since the RCAT product remains attached to the original circular template, the method may be used in combination with DNA microarrays (see Ch.24 for DNA arrays).

In exponential RCAT (E-RCAT) a second primer is used that binds to the opposite strand—in other words it binds to the newly made linear strand. When this primer binds to neighboring tandem copies of the target sequence elongation results in strand displacement. This creates single-strand branches that in turn can bind primer number 1 and so be converted to double-stranded DNA. Alternate extension using two primers results in multiple branching of the amplified DNA (Fig. 23.27). The exponential version of RCAT can manufacture 1012 copies of each original circle in an hour and can detect a single target molecule. It is thus superior to PCR in both aspects.

rolling circle amplification technology (RCAT) Method based on rolling circle replication that uses DNA polymerase to amplify target DNA at normal temperatures

Primer 1

New DNA from P1

Primer 1

Elongation of P1

New DNA from P1

Elongation OF P2

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