Screening of 16S rDNA gene banks

Prescreening with ARDRA (e.g. Martinez-Murcia et al. 1995)

Principle:

ARDRA (Amplified rDNA Restriction Analysis) is used to estimate the diversity within a gene bank without sequencing. The 16S rDNA insert of the clones (n > 50) is digested by restriction endo-nucleases which bind at specific target sites on double strand DNA and split the helix. According to the locations of the binding sites, the restriction fragments have variable lengths and can be separated by polyamide gel-electrophoresis (PAGE). The restriction fragments produce a band pattern which is specific to the sequence of the digested DNA. Restriction endo-nucleases with short target sequences (e.g. 4 bp) produce a large number of bands.

Solutions:

1 x buffer C (Promega, Madison, USA)

Restriction:

1 x buffer C

7.5 U Hae III

7.5 U Rsa I

(Continue with screening of 16S rDNA gene banks methods on next page)



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