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)

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.

T-RFLP analysis measures length & quantity of labeled (terminal) restriction fragments.

Unlabeled restriction fragments ignored.

Amplicons purified to remove reaction buffer, unincorporated primers & dNTPs.

Agarose gel electrophoresis confirm amplicon length (~880-940 bp).

Measure concentration of PCR product (1 l) using nanodrop spectrophotometer.

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.

Instrument uses size standards with different fluorescent tag to estimate size of labeled restriction fragments.

Estimates may differ from true length by several nucleotides.

mixed templates: PCR primers:Fam-labeled 8-27F

unlabeled 926-907R

PCR:

AluI AluI

AluI

AluI

AluI

AluI

Digest with AluI

Fam-labeled AluI restriction fragments

Capillary electrophoresis:F

luor

esce

nce

inte

nsi

ty

Fragment length (nucleotides)

Prevotella

Veillonella

Leptotrichiasp. HKU24

Crohn’s patient #10 Msp 2009

Capnocytophaga

Crohn’s patient #10 Msp 2011

Streptococcus

Neisseria

Prevotella

Streptococcus

Veillonella

Leptotrichiasp. A39FD