Techniques to manipulate DNA
Phenol/chloroform extraction
Ethanol precipitation
Concentration determination
Gel Electrophoresis
DNA hybridization
Southern Blotting
Melting temperatures
PCR
BRCA3EcoRI EcoRI
NIH
Example,Is the BRCA3 gene involved in
Suppressing breast cancer?
BRCA1
BRCA2
BRCA3
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenol
Chloro
ETOH ppt
Spin
The,Hyperchromic Shift”
2
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenol
Chloro
ETOH ppt
Spin
Agarose Gel Electrophoresis
_ +
3
Sample Well
25 ng 1 kb ladder
0.8% Agarose
1
2
4
6
810
12
kb Agarose Gel Electrophoresis
Sybergold Detection Limit,~0.2-0.5 ng DNA
EtBr Detection Limit,~5-10 ng DNA
(Not in handout)
Log (MW) is linearly related to mobility
4100 bp
Low % has no
Resolving power
at low MW
1 kb
10 kb
High % has no
resolving power
at high MW
Separation Range Vs,% Agarose
5Low Geling Agarose 4-6 0.02-0.4
TEMED
(N,N,N,N-tetramethylenediamine)
7
PolyAcrylamide Gels
8
DNA topology affects mobility of DNA
9
PFGE can resolve large DNA fragments
10
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenol
Chloro
ETOH ppt
Spin
Helix Formation,Nucleation and Propagation
11
A-T
A-T
G-C
Helix Melting
12
What parameters affect the
Tm?
(Tm is defined as the temp at
which half the molecules
are single-stranded)
Tm depends on DNA length
13
%
dsDN
A
Sigmoidal
Curve:
Highly
Cooperative
Melting!
Effect of % GC Content on Tm
14
Effect of [Salt] on Tm
15
i ) F o r o li g o s 4 - 1 8 nu cl e ot i d e s
T
m
= ( # A - T b p ) 2 ? C + (# G - C b p ) 4 ? C
i i ) F o r 1 4?7 0 n u cl e o t i d es
0, 0 1 M < [ N a + ] < 0,4 M,a n d 3 0 < %G C < 7 5
T
m
= 8 1,5 + 1 6,6 l o g 1 0 [N a +] + 0, 4 1 ( % G + C ) 6 0 0 / N – P
w h e r e N = # n u c l e o t i de s,P = % m is m at c h e s
Predicting melting temperatures
(see lecture notes)
Kinetics of Hybridization:
Do I have time for a soda or a
week-end in New York City?
Cot Curves,Hybridization vs,Complexity
16
A Bi-Phasic Cot Curve
17
5 kb Repeats (20% total)
100 kb fragment
Repeat DNA = 5 kb
Longest non-repeat DNA = 100kb - 20 kb = 80 kb
Difference in complexity = 80/5 = 16
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenol
Chloro
ETOH ppt
Spin
Results of Southern Blot
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIH
BRCA3EcoRI EcoRI
NIH
94 ?C
(30 s)
40-60 ?C
(30 s)
72 ?C
(30-60 s/kb)
Melt Anneal Extend
The Polymerase Chain Reaction (PCR)
1820-30X
94 ?C
(30 s)
40-60 ?C
(30 s)
72 ?C
(30-60 s/kb)
Melt Anneal Extend
Cycle #2
NIH20-30X
Results of PCR #1
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIHPrimer Set #1
Results of PCR#5
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIHPrimer Set #5
PCR mutagenesis
EcoRIHindIII
Isolate products,mix
Amplify
digest,subclone sequence 20
PvuII
Linear
Amplification
Geometric
Amplification
19
hsp70
EcoRI EcoRI
NIH
Example,Is heat shock factor bound to
promoters before heat shock?
EcoRI
HSE
HSF
Optimal separation of fragments > 2 kb
requires low voltage
0.5 10 V/cm
6