Презентация на тему PCR and sequence

where the strands separate (DNA melts).
medlib.med.utah.edu/block2/ biochem/
ssDNA

Unstacked bases
(random orientation)
absorb more light
than neatly stacked
(oriented) base-pairs

Melting curve.

The temperature
at which DNA is half unfolded

Tm (melting temp)

Tm is a measure of the stability of dsDNA under a given set of conditions

Hypochromic
Shift

Composition :
higher the GC content, the higher the

2. Ionic Strength :
as the ionic strength increases, so does Tm.
Double helical DNA is stabilized by cations.

Divalent cations (Mg2+) are more effective
than monovalent cations (NA+ or K+).

3. Organic Solvents –
formamide for instance
lowers the Tm by weakening
the hydrophobic interactions.

more STABLE
When contains many GC

T in hybridization)
http://www.biology.arizona.edu/molecular_bio/problem_sets/m/graphics/05ta.gif

SOLUTION

C



(hybridization
is more specific)

use for specific DNA detection
Specific probe self-anneals to target

Only problem – DNA is invisible

How to visualize DNA?

Radioactively

Fluorescently

I (polymerase activity)
Will work without DNAse,
as there are

at the University of Oxford
.

Et Br
VIZUALIZED by P32

by each different blot type?

carrying a particular DNA clone
http://amiga1.med.miami.edu/Medical/PDF-Files/Lecture10.pdf

known proteins only)
http://amiga1.med.miami.edu/Medical/PDF-Files/Lecture10.pdf

of unique sequences it corresponds to (5 in one

can be used when some of the related genomic sequences
are unknown, or known only in a related species.

Up to 1010 degeneracy is tolerated

to biotin,
is hybridized to
a denatured chromosome preparation.

http://www.childsdoc.org/spring2000/missinggenes.asp

of association
of yet observed for noncovalent binding
of

Streptavidin is a protein.
1 mole of SA binds 4 moles Bio

BIOTIN is a vitamin B
(small thing)

Biotin could be added to nucleotide
and incorporated into the probe

(67 kD protein
from Streptococcus avidinii)

Avidin could be
conjugated with fluorophore

visualize mRNA expression in tissues and cells. 
Encephalin gene

www.omrf.org/OMRF/ Core/InSitu.asp

The HuC transcript is expressed specifically
in the nervous system of this E18 mouse

www.infobiogen.fr/.../
Metaphase FISH analysis
using the BAC probe RP11-104M2

-- RED

annealing
DNA elongation
Nobel Prize in Chemistry 1993,
at age

Kary Mullis
(invented PCR in 1983)

PhD
"The Cosmological Significance
of Time Reversal,"

Biochemistry from U.C. Berkeley

software

Try for equal Tm for both primers

PCR is often an issue
www.biotechlab.nwu.edu/ pe/Sld022.htm
mkM

Yield and High Fidelity
are mutually exclusive

amplified
www.biotechlab.nwu.edu/ pe/Sld022.htm

PCR reaction

PCR
5%
D
M
S
O
D
M
S
O
+
G
L
Y
M
A
R
K
E
R
Decrease in Mg concentraton

for PCR,
is still the most popular.
-- high

-- generates PCR products
with single A overhangs on the 3´-ends
(Suitable for TOPO-cloning)

“Topo” cloning system (Invitrogen)

Half-life at 95C is 1.6 hours

in the presence of Mn2+ ions.
DNA-dependent DNA-polymerase activity

The fragment should be ideally smaller 1 kb.

Mn 2+

Mg 2+

Pyrococcus furiosus).
approx.1 / 2, 000,000 nucleotides before making

can tolerate temperatures exceeding 95°C,
enabling it to PCR amplify GC-rich targets.

more expensive

polymerase
Very termostable: Half-life at 95 C is approximately

Vent error rate is intermediate between Taq and Pfu.

2-5 x 10-5 errors/bp

3'->5' exonuclease activity presents

Other polymerases:

Deep Vent (Pyrococcus species GB-D) (New England Biolabs)
New England Biolabs claims fidelity is equal to or greater than that of Vent.

Replinase (Thermus flavis) ?1.03 x 10-4 errors/base

Taq
is the main polymerase in the reaction,
a proofreading

22-24 kb PCR products are achieved on
Qiagen and Eppendorf PCR mixes

Taq+ Pwo
(Pyrococcus woesei) ;
Pwo is very stable,
2 hrs at 100 C

(Nobel prize 1980 with Paul Berg and Walter Gilbert)

that combines the PCR and actual sequencing

System
Promoter system -> expression vector
Properties of product -> stability
Production

foreign protein