免疫学信息网 Techniques to manipulate DNA Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis

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Techniques to manipulate DNA

Phenol/chloroform extractionEthanol precipitationConcentration determinationGel ElectrophoresisDNA hybridizationSouthern BlottingMelting temperaturesPCR


BRCA3EcoRI EcoRI

NIH

Example: Is the BRCA3 gene involved in Suppressing breast cancer?

BRCA1

BRCA2

BRCA3


Paper Towels

Southern Blotting

1

tissue

SDSProtKPhenolChloro

ETOH ppt

Spin


The “Hyperchromic Shift”

2


Paper Towels

Southern Blotting

1

tissue


ETOH ppt

Spin


Agarose Gel Electrophoresis

_ +

3


Sample Well 25 ng 1 kb ladder0.8% Agarose

1

2

4

681012kb

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 noResolving powerat low MW

1 kb

10 kb

High % has noresolving powerat 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


ETOH ppt

Spin


Helix Formation: Nucleation and Propagation

11


A-T

A-T

G-C

Helix Melting

12


What parameters affect theTm?

(Tm is defined as the temp at which half the molecules

are single-stranded)


Tm depends on DNA length

13

% d

sDN

ASigmoidalCurve:HighlyCooperativeMelting!


Effect of % GC Content on Tm

14


Effect of [Salt] on Tm

15


i) For oligos 4 - 18 nucleotides

Tm = (#A-T bp) 2 C + (#G-C bp) 4C

ii) For 14 – 70 nucleotides

0.01 M < [Na+] < 0.4 M, and 30 < %GC < 75

Tm = 81.5 + 16.6 log10[Na+] + 0.41 (% G + C)– 600/N – P

where N = # nucleotides, P = %mismatches

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 kbLongest non-repeat DNA = 100kb - 20 kb = 80 kbDifference in complexity = 80/5 = 16


Paper Towels

Southern Blotting

1

tissue


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


BRCA3EcoRI EcoRI

NIHPrimer Set #1


Results of PCR#5


BRCA3EcoRI EcoRI

NIHPrimer Set #5


PCR mutagenesis

EcoRIHindIII

Isolate products, mix

Amplify

digest, subclone sequence 20

PvuII



Linear Amplification

GeometricAmplification

19


hsp70

EcoRI EcoRI

NIH

Example: Is heat shock factor bound to promoters before heat shock?

EcoRI

HSE

HSF


Optimal separation of fragments > 2 kbrequires low voltage

0.5 10 V/cm

6

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免疫学信息网 Techniques to manipulate DNA Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis