wzwlabJR-stamp

PresenterPresentation Notes DNA , . RNA . RNA .

(dATP, dTTP, dGTP, dCTP)

:

3-5

.

PresenterPresentation Notes DNA , , 2 . , , . 3 -5 . .

DNA

PresenterPresentation Notes DNA , , . .

=

PresenterPresentation Notes DNA . DNA . . , . . .

(ATP, GTP, CTP, UTP)

PresenterPresentation Notes DNA , . , pH . DNA , . DNA , . . (melting temperature) 50% . .

PresenterPresentation Notes DNA . , , , . DNA. DNA DNA . : DNA. 5-3. RNA , 3 . DNA , DNA. , . DNA. DNA DNA . . .

DN

DNA

,

.

PresenterPresentation Notes DNA . . . EcoRI E. coli. DNA . DNA DNA. . . .

,

DNA

DNA

(

)

DNA

DNA

(Pulse Field Gel Electrophoresis, PFGE )

DNA

DNA markers

Methylene

blue

SybrGreen

I, II

DNA >40 kb

(pulses)

DNA

DNA

DNA

Gram

Gram

1

DNA-DNA

DNA-RNA

RNA-RNA

(probe)

(background)

100%

.

,

probe

,

RNA

RNA

RNA

probe

HLA

HPV

,

(

)

(

)

-

18-30

,

DNA

,

:

:

DNA

dNTPs

DNA,

DNA

:

:

MgCl

DNA

:

:

()

DNA

ELISA

ELISA

probe

PCR

-ds

DNA

.

DNA-

2

mRNA

RNA

DNA

,

RNA

DNA (cDNA)

PCR

Thermus thermophilus

5 10 15 20 30 40 500

NTC

Plateau

PCR (

)

Real-Time PCR

PCR

PCR

:

:

DNA

RNA

FITC

Red 640

FRET

P

Amplicon

PresenterPresentation NotesThere are currently a number of different probe formats which are used in real time PCR all of which use which use fluorescence resonance energy transfer. Probably the common of these are the Taqman probes however we have settled on using adjacent hybridisation probes. Unlike taqman which only uses a single probe this format uses 2 probes. The way in which these probes work is that the 2 probes align on specific PCR product. This allows the energy from the fluorophore on the first probe to be transferred to the flourophore on the 2nd probe. It is this energy transfer and subsequent fluorescence which is monitored by the instrument. Essentially as the PCR product accumulates the fluorescence which is emited by the reaction mix increases.

ss DNA

Taq polymerase

ds DNA

Oligo

1: Fluorescein Oligo

2: LC640

Reporter fluorophore Quencher

Taq

DNA

F

R

R Q

PCR

probe

-

RQ

probe

RQ

probe

RQ

TaqMan

probes

.

quencher

multiplex PCR

DNA

Quencher

R Q

R

Q

QR

probes

Cro

ssin

g Po

int (

Cyc

les)

log (

)

(

)

1.

log

(F2/

F1)

log

(F2/

F1)

Threshold

Crossingpoint Standard

QS 1 21.4 1x105 cop/l 1.15x105 cop/l QS 2 24.9 1x104 cop/l 1.26x104 cop/l QS 3 28.7 1x103 cop/l 0.85x103 cop/l QS 4 31.9 1x102 cop/l 1.12x102 cop/l QS 5 35.8 1x101 cop/l 0.81x101 cop/l

26.1 6.21x10

3 cop/l

H S V 2 H S V 1

M e ltin g C u rve A n a ly s is

H S V 1

H S V 2

5 5 oC

H S V 1

H S V 2

7 3 oC

H S V 1

H S V 2

6 7 oC

DNA pol

HSV

HSV-1

HSV-2

Hybridisation

probes (

HSV-1)

100 % Amplicon

Primers

HSV 1 & 2

RNA

109

2

DNA

RNA

(10-50

)

M. tuberculosis

Chl. Trachomatis

N. gonorrhoeae

ddATP

ddGTPddGTP

ddCTPddCTP

ddTTP

/

,

,

(, ),

probe

.

probe

400.000/20

m

RNA

mRNA

mRNA

mRNA

cDNA

cDNA

cDNA

microarray

cDNA

laser

:

:

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