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Techniques to manipulate DNA Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis DNA hybridization Southern Blotting Melting temperatures PCR


Example: Is the BRCA3 gene involved in Suppressing breast cancer? BRCA1 BRCA3 BRCA2

EcoRI

BRCA3

EcoRI

NIH


Southern Blotting ETOH ppt Spin

Phenol Chloro

ProtK

SDS

tissue

Paper Towels

1


The “Hyperchromic Shift”

2


Southern Blotting ETOH ppt Spin

Phenol Chloro

ProtK

SDS

tissue

Paper Towels

1


Agarose Gel Electrophoresis

_

+

3


Sample Well 25 ng 1 kb ladder 0.8% Agarose

kb 12 10 8 6 4

Agarose Gel Electrophoresis Sybergold Detection Limit: ~0.2-0.5 ng DNA EtBr

Detection Limit: ~5-10

ng DNA

2

1

(Not in handout)


Log (MW) is linearly related to mobility High % has no resolving power at high MW 10 kb

1 kb

Low % has no Resolving power at low MW 100 bp

4


Separation Range Vs. % Agarose

Low Geling Agarose 4-6

0.02-0.4

5


TEMED (N,N,N,N-tetramethylenediamine)

7


PolyAcrylamide Gels

8


DNA topology affects mobility of DNA

9


PFGE can resolve large DNA fragments

10


Southern Blotting ETOH ppt Spin

Phenol Chloro

ProtK

SDS

tissue

Paper Towels

1


Helix Formation: Nucleation and Propagation

11


Helix Melting

A-T G-C A-T

12


What parameters affect the Tm? (Tm is defined as the temp at which half the molecules are single-stranded)


% dsDNA

Tm depends on DNA length Sigmoidal Curve: Highly Cooperative Melting!

13


Effect of % GC Content on Tm

14


Effect of [Salt] on Tm

15


Predicting melting temperatures i) For oligos 4 - 18 nucleotides Tm = (#A-T bp) 2 °Χ + (#Γ −Χ βπ) 4°Χ

ιι) Φορ 14 – 70 νυχλεοτιδεσ 0.01 Μ < [Να+] < 0.4 Μ, ανδ 30 < %Γ Χ < 75 Τµ = 81.5 + 16.6 λογ10[Να+] + 0.41 (% Γ + Χ)– 600/Ν – Π ωηερε Ν = # νυχλεοτιδεσ, Π= %µ ισµ ατχηεσ

(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 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

17


Southern Blotting ETOH ppt Spin

Phenol Chloro

ProtK

SDS

tissue

Paper Towels

1


Results of Southern Blot EcoRI

EcoRI

BRCA3

Control Tissue

Cancer Tissue

NIH


EcoRI

BRCA3

EcoRI

NIH


The Polymerase Chain Reaction (PCR) 40-60 째C (30 s)

72 째C (30-60 s/kb)

Anneal

Extend

94 째C (30 s)

Melt

20-30X

18


Cycle #2 40-60 째C (30 s)

72 째C (30-60 s/kb)

Anneal

Extend

94 째C (30 s)

Melt

20-30X

NIH


Results of PCR #1 EcoRI

EcoRI

BRCA3

Control Tissue

Cancer Tissue

Primer Set #1

NIH


Results of PCR#5 EcoRI

EcoRI

BRCA3

Control Tissue

Cancer Tissue

Primer Set #5

NIH


PCR mutagenesis HindIII

PvuII

EcoRI

Isolate products, mix

Amplify

digest, subclone sequence

20


Linear Amplification

Geometric Amplification

19


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

HSF HSE

EcoRI

EcoRI

hsp70

NIH


Optimal separation of fragments > 2 kb requires low voltage

0.5 10 V/cm

6


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