1

Purifying DNA & RNA

Source

Amounts & Purity

Damage or Loss

2

Bind DNA

Cells

Pure DNA

Extract

Remove junk

3

DNA, RNASolution

DenaturedProtein

Phenol

CellExtract

Shake Spin

1. Remove high MW junk

4

(to 70%)

SPIN (fast)

Add

and salt

2. Remove low MW junkand concentrate

5

BIND + SALT(aided by alcohol)

ELUTE - NO SALT(just add water)

Or Bind DNA

6

EXTRACT INHigh Salt

SILICAPARTICLES

WATER

DNA

High SaltWash

7

Purifying one type of DNAaway from other DNA molecules

-Plasmids from bacterial chromosomal DNA(Form)

- phage DNA(Location)

-Restriction fragments, PCR products(Size)

8

SDS, alkali

9

10

alkali

neutralize

11

RNA PURIFICATIONLyse & denature proteins FAST Acidic phenol

Or bind glass/silica

Small RNAs?

12

13

14

15

A? B? C?M

16

Oligo-dT beads for polyA+ mRNA

17

High Salt

18

19

20

Chemical Synthesisof oligonucleotides

Uses?

Block and unblock sequentiallyso that only one nucleotide addsat a time

21

Phosphoramidite

5’

3’

Protected amino groups

*

*

22

Couple

Growingchain

Blocked 5’-OH

23

Unblock 1st “nucleotide”

24

Add and activate next “nucleotide”

25

Couple

26

Product99%

27

1% Cap

28

29

1st nuc on bead.(blocked at 5’-OH)

unblock

couple

cap

Remove frombead

Purify

3’- 5’

Add next nuc. (blocked 5’)

De-protect

Unblock 5’-OH

30

PNA

RNA harderDNA variants like

31

32

33

If DNA is too large for conventional electrophoresis….

34

Pulsed-field electrophoresis

35

36

37

Polyacrylamide Gels can resolve smallDNAs differing inlength by onenucleotide

38

Dideoxy sequencing converts sequence Information (A, C, G, T) into size differences

e.g. if a DNA has T residues at positions2, 5, 13, 16… this can be converted intoa set of DNAs of length n+ 2, 5, 13, 16..(which can be measured by denaturingpolyacrylamide gel electrophoresis)

39

40

ddCTP

41

ddATPdCTPdGTPdTTP

dATP

LABEL

42

2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

43

44

2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

n +

45

46

2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

47

ddAddCddGddT

48

49

50

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?

51

Nucleic acid hybridization

Key to life and almost everyprocedure in molecular biology

52

53

54

55

56

Hybridize to DNAon blot

57

Recognized by specific Ab

Recognized by (strept)avidin

58

RNA Probe

59

60

NorthernRNA blot

61

COLONYHYBRIDIZATION

62

DNA (chromosome) in situ

FISH

63

64

65

66

RNA in situ with non-radioactive probe