mcgill - 300d2 - polymerase chain reaction in detail

8/6/2019 McGill - 300D2 - Polymerase Chain Reaction in Detail

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Quantification of nucleic acids PCR is a highly sensitive quantification method other techniques: Dot Blot, Southern Blot etcConventional PCR quantification methods:

1. Many reaction tubes with different amount of template (need aninternal control)

2. Amplification products are detected by gel electrophoresis andstained with EtBr.

3. Southern Blotting*Very laborious


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

The quantitative information in a PCR comes from the cycles where the amountof DNA grows logarithmically (before it reaches a plateau level).

The log-linear phase appears at higher cycle numbers as the number oftemplate copies in the reaction decreases.


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Quantification

*Note that the

log-linear phase

is only 4 to 5

cycles long


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The samples are continuously monitoredduring the amplification at each cycle (dsDNA

binding dye)

The log-linear region is easily identified as thefluorescence data appears on the computer

screen

A single reaction takes the place of manyreactions


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SYBR Green I is a ds DNA minor groove binding dyeSYBR Green I exhibits little

fluorescence in the unbound

state

SYBR Green I exhibits 1000Xmore fluorescence in the bound

state


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Standard 6 x 105 copies 5 Standard 6 x 104 copies 4 Standard 6 x 103 copies 3 Standard 6 x 102 copies 2 unknown sample U unknown sample U

5 4 U 3 U 2

More templatemolecules is initially

present, the fewer the

# cycles to get to the

THRESHOLD

CYCLE

cycle at which

fluorescence is

statistically

significant above

background

Inversely

proportional to

log of initial

template #


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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


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l

og

(F2/F1)

Target

Cycle #

log

(F2/F1)

106 105 104 103 copies

Cycle #

#

Cycles

log (copy number)


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Gel electrophoresis

(EtBr Staining)

Melting Curve analysis


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At end of PCR the To is slowlyraised and fluorescence is

measured at frequent intervals

The fluorescence of the SYBRGreen I dye declines sharply as

the dsDNA are denatured

Fluorescence is plotted againstincreasing temperature

The inflection point of thecurve gives the Tm of the PCR

product (see red/blue vertical

lines)

Each dsDNA fragment has aTm ( To at which 50% of the

DNA is ss and 50% ds)


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Each DNA fragment has its own Tm which is mainly determined by GC content

and length of the fragment

mcgill - 300d2 - polymerase chain reaction in detail

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