Quantification of nucleic acids PCR is a highly sensitive quantification method

other techniques: Dot Blot, Southern Blot etc Conventional PCR quantification methods:
1. Many reaction tubes with different amount of template (need an internal control)

2. Amplification products are detected by gel electrophoresis and stained with EtBr. 3. Southern Blotting *Very laborious

The quantitative information in a PCR comes from the cycles where the amount of DNA grows logarithmically (before it reaches a plateau level). The log-linear phase appears at higher cycle numbers as the number of template copies in the reaction decreases.

Quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle.

Quantification

*Note that the log-linear phase is only 4 to 5 cycles long

 The samples are continuously monitored during the amplification at each cycle (dsDNA binding dye) The log-linear region is easily identified as the fluorescence data appears on the computer screen A single reaction takes the place of many reactions

SYBR Green I is a ds DNA minor groove binding dye SYBR Green I exhibits little fluorescence in the unbound state SYBR Green I exhibits 1000X more fluorescence in the bound state

 More template molecules is initially present, the fewer the # cycles to get to the THRESHOLD CYCLE

cycle at which fluorescence is statistically significant above background

U3

2
5 4 3 2 U U

Inversely proportional to log of initial template #

Standard 6 x 105 copies Standard 6 x 104 copies Standard 6 x 103 copies Standard 6 x 102 copies unknown sample unknown sample

After completion of PCR, calculate concentration by plotting log (Fluorescence) vs cycle # and setting a baseline (crossing line) which identifies the cycle at which the log-linear signal can be distinguished from the background for each sample. Each crossing point on the baseline is plotted against the log (copy number) to produce a standard curve

Target log (F2/F1)

Cycle #
106 105 104 103 copies

log (F2/F1)

Cycle #

# Cycles log (copy number)

Gel electrophoresis (EtBr Staining)

Melting Curve analysis

 At end of PCR the To is slowly raised and fluorescence is measured at frequent intervals The fluorescence of the SYBR Green I dye declines sharply as the dsDNA are denatured Fluorescence is plotted against increasing temperature The inflection point of the curve gives the Tm of the PCR product (see red/blue vertical lines) Each dsDNA fragment has a Tm ( To at which 50% of the DNA is ss and 50% ds)

Each DNA fragment has its own Tm which is mainly determined by GC content and length of the fragment