Purifying DNA & RNA
Source
Amounts & Purity
Damage or Loss
1
Cells
Extract
Remove junk
Pure DNA
Bind DNA
2
1. Remove high MW junk
DNA, RNA
Solution
Denatured
Protein
Phenol
Cell
Extract
Shake
Spin
3
2. Remove low MW junk
and concentrate
Add
SPIN (fast)
(to 70%)
and salt
4
Or Bind DNA
BIND + SALT
(aided by alcohol)
ELUTE - NO SALT
(just add water)
5
EXTRACT IN
High Salt
High Salt
Wash
WATER
SILICA
PARTICLES
DNA
6
Purifying one type of DNA
away from other DNA molecules
-Plasmids from bacterial chromosomal DNA
(Form)
- phage DNA
(Location)
-Restriction fragments, PCR products
(Size)
7
SDS, alkali
8
9
alkali
neutralize
10
RNA PURIFICATION
Lyse & denature proteins FAST
Acidic phenol
Or bind glass/silica
Small RNAs?
11
12
13
14
M
A?
B?
C?
15
Oligo-dT beads for polyA+ mRNA
16
High Salt
17
18
19
Chemical Synthesis
of oligonucleotides
Uses?
Block and unblock sequentially
so that only one nucleotide adds
at a time
20
Phosphoramidite
*
Protected amino
groups
5’
3’
*
21
Blocked 5’-OH
Couple
Growing
chain
22
Unblock 1st “nucleotide”
23
Add and activate next “nucleotide”
24
Couple
25
Product
99%
26
1%
Cap
27
28
3’- 5’
1st nuc on bead.
(blocked at 5’-OH)
unblock
Add next nuc. (blocked 5’)
Remove from
bead
couple
De-protect
Unblock 5’-OH
Purify
cap
29
RNA harder
DNA variants like
PNA
30
31
32
If DNA is too large for conventional electrophoresis….
33
Pulsed-field electrophoresis
34
35
36
Polyacrylamide Gels
can resolve small
DNAs differing in
length by one
nucleotide
37
Dideoxy sequencing converts sequence
Information (A, C, G, T) into size differences
e.g. if a DNA has T residues at positions
2, 5, 13, 16… this can be converted into
a set of DNAs of length n+ 2, 5, 13, 16..
(which can be measured by denaturing
polyacrylamide gel electrophoresis)
38
39
ddCTP
40
dATP
ddATP
dCTP
dGTP
dTTP
LABEL
41
ddA
2,5,13,16
ddC
9,10,15,19
ddG
ddT
1,4,7,8,12,14,20
3,6,11,17,18
42
43
n+
ddA
2,5,13,16
ddC
9,10,15,19
ddG 1,4,7,8,12,14,20
ddT
3,6,11,17,18
44
45
ddA
2,5,13,16
ddC
9,10,15,19
ddG
ddT
1,4,7,8,12,14,20
3,6,11,17,18
46
ddA
ddC
ddG
ddT
47
48
49
What do you need to sequence DNA?
Where do the reagents come from?
Must the DNA be pure? How much is needed
How much good sequence can you obtain?
50
Nucleic acid hybridization
Key to life and almost every
procedure in molecular biology
51
52
53
54
55
Hybridize to DNA
on blot
56
Recognized by specific Ab
Recognized by (strept)avidin57
RNA Probe
58
59
Northern
RNA blot
60
COLONY
HYBRIDIZATION
61
DNA (chromosome) in situ
FISH
62
63
64
65
RNA in situ with non-radioactive probe
66