Post on 20-Jan-2016
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Purifying DNA & RNA
Source
Amounts & Purity
Damage or Loss
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Bind DNA
Cells
Pure DNA
Extract
Remove junk
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DNA, RNASolution
DenaturedProtein
Phenol
CellExtract
Shake Spin
1. Remove high MW junk
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(to 70%)
SPIN (fast)
Add
and salt
2. Remove low MW junkand concentrate
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BIND + SALT(aided by alcohol)
ELUTE - NO SALT(just add water)
Or Bind DNA
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EXTRACT INHigh Salt
SILICAPARTICLES
WATER
DNA
High SaltWash
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Purifying one type of DNAaway from other DNA molecules
-Plasmids from bacterial chromosomal DNA(Form)
- phage DNA(Location)
-Restriction fragments, PCR products(Size)
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SDS, alkali
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alkali
neutralize
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RNA PURIFICATIONLyse & denature proteins FAST Acidic phenol
Or bind glass/silica
Small RNAs?
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A? B? C?M
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Oligo-dT beads for polyA+ mRNA
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High Salt
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Chemical Synthesisof oligonucleotides
Uses?
Block and unblock sequentiallyso that only one nucleotide addsat a time
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Phosphoramidite
5’
3’
Protected amino groups
*
*
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Couple
Growingchain
Blocked 5’-OH
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Unblock 1st “nucleotide”
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Add and activate next “nucleotide”
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Couple
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Product99%
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1% Cap
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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
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PNA
RNA harderDNA variants like
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If DNA is too large for conventional electrophoresis….
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Pulsed-field electrophoresis
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Polyacrylamide Gels can resolve smallDNAs differing inlength by onenucleotide
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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)
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ddCTP
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ddATPdCTPdGTPdTTP
dATP
LABEL
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2,5,13,16
9,10,15,19
1,4,7,8,12,14,20
3,6,11,17,18
ddA
ddC
ddG
ddT
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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 +
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2,5,13,16
9,10,15,19
1,4,7,8,12,14,20
3,6,11,17,18
ddA
ddC
ddG
ddT
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ddAddCddGddT
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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?
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Nucleic acid hybridization
Key to life and almost everyprocedure in molecular biology
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Hybridize to DNAon blot
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Recognized by specific Ab
Recognized by (strept)avidin
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RNA Probe
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NorthernRNA blot
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COLONYHYBRIDIZATION
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DNA (chromosome) in situ
FISH
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RNA in situ with non-radioactive probe