PCR and sequence

strands separate (DNA melts).
medlib.med.utah.edu/block2/ biochem/
ssDNA
H-bonds between basepairs

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

higher the GC content, the higher the Tm.
2. Ionic Strength

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.

contains many GC

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

more specific)

specific DNA detection
Specific probe self-anneals to target DNA
Only problem –

How to visualize DNA?

Radioactively

Fluorescently

activity)
Will work without DNAse,
as there are always nicks in

University of Oxford
.

by P32

different blot type?

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

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

sequences it corresponds to (5 in one of the examples

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

is hybridized to
a denatured chromosome preparation.

An avidin-bound fluorescent

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

of yet observed for noncovalent binding
of a protein and

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

expression in tissues and cells. 
Encephalin gene expression
in the

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

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

FISH analysis
using the BAC probe RP11-104M2
labeled with FITC

Nobel Prize in Chemistry 1993,
at age 48
Kary Mullis
(invented

PhD
"The Cosmological Significance
of Time Reversal,"

Biochemistry from U.C. Berkeley

primers

Fidelity
are mutually exclusive

Decrease in Mg concentraton

is still the most popular.
-- high processivity
-- is

-- 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

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

The fragment should be ideally smaller 1 kb.

Mn 2+

Mg 2+

approx.1 / 2, 000,000 nucleotides before making an error.

In

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

more expensive

termostable: Half-life at 95 C is approximately 7 hours
Vent

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

main polymerase in the reaction,
a proofreading polymerase (3' to

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

1980 with Paul Berg and Walter Gilbert)

the PCR and actual sequencing

-> expression vector
Properties of product -> stability
Production level