Inverse PCR

Another approach that uses incomplete sequence information to amplify a target gene is inverse PCR. In this case a sequence of part of a long DNA molecule, say a chromosome, is known. The objective is to extend the analysis along the DNA molecule into the unknown regions. To synthesize the primers for PCR, the unknown target sequence must be flanked by two regions of known sequence. The present situation is exactly the opposite of that. To circumvent this problem, the target molecule of DNA is converted into a circle. Going around a circle brings you back to the beginning. In effect, even though, only one small stretch of sequence is known, the circular form allows you to have that one region on both sides of the target sequence.

A restriction enzyme, usually one that recognizes a six-base sequence, is used to make the circle. This enzyme must not cut into the known sequence, therefore, eventually, this enzyme will cut either upstream or downstream from the known region.

inverse PCR Method for using PCR to amplify unknown sequences by circularizing the template molecule

Step 1: making the template

1 -, Left side Known sequence Right side

Recognition site for restriction enzyme cut with

RESTRICTION ENZYME; LIGATE ENDS

FIGURE 23.09 Inverse PCR

Inverse PCR allows unknown sequences to be am plified by PCR provided that they are located next to DNA whose sequence is already known. The DNA is cut with a restriction enzyme that does not cut within the region of known sequence, as shown in Step I. Th is generates a fragment of DNA containing the known sequence flanked by two regions of unknown sequence. Since the fragment has two matching sticky ends, it may be easily circularized by DNA ligase. Finally, PCR is performed on the circular fragments of DNA (Step 2). Two primers are used that face outwards from the known DNA sequence. PCR amplification gives a single linear product that includes unknown DNA from both left and right sides. This PCR product can now be cloned and/or sequenced.

PCR primers

Sticky ends join

Sticky ends join

Step 2: run pcr reaction

Short segment of known sequence

Sticky ends

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