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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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