1. Laboratory: Unit 4: PCR for T-RFLP (pages 83-84) Lecture: Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis In-Class Writing: peer review editorial (xlii & 96) Read: manual pages 71-100, 213 Hand In: flow chart 4; editorial draft Due Next Class: editorial (page 96)

2. T-RFLP analysis: PCR of 16S rRNA genes. One fluorescently labeled primer & one unlabeled primer  one end of amplicon labeled. PCR product contains mixture of different amplicons (16S rRNA genes from each bacterial species.

3. T-RFLP analysis measures length & quantity of labeled (terminal) restriction fragments. Unlabeled restriction fragments ignored. Amplicons purified to remove reaction buffer, unincorporated primers & dNTPs.

4. Agarose gel electrophoresis  confirm amplicon length (~880-940 bp). Measure concentration of PCR product (1  l) using nanodrop spectrophotometer.

5. Digest 100 ng of PCR product with: RsaI (GT/AC) MspI (C/CGG) Digested DNAs analyzed by capillary electrophoresis. Separates DNA molecules that differ by 1 nucleotide.

6. Instrument uses size standards with different fluorescent tag to estimate size of labeled restriction fragments. Estimates may differ from true length by several nucleotides.

7. mixed templates:PCR primers: Fam-labeled 8-27F unlabeled 926-907R PCR:

8. AluI Digest with AluI Fam-labeled AluI restriction fragments

9. Capillary electrophoresis: Fluorescence intensity Fragment length (nucleotides)