1. CENTRAL DOGMA OF MOLECULAR BIOLOGY 2. NUCLEIC ACID PREPARATION & APPLICATIONS 3. FUNDAMENTAL STEPS IN DNA PURIFICATION 4. ANALYSIS OF NUCLEIC ACIDS 5. STORAGE CONDITIONS

2. The Central Dogma of Molecular Biology
Replication
Transcription
Translation
mRNA
non-coding RNA (rRNA, tRNA, siRNA, etc.)

3. Deoxyribonucleotide
Ribonucleotide

4. NUCLEIC ACID PREPARATION
Application?
Amplification methods (PCR, LCR)
Restriction enzyme digest
Hybridization methods (Southern analysis)
Sequencing
RNA
Amplification methods (RT-PCR)
Hybridization methods (Northern analysis)
DNA
The very first DNA isolation was done by a Swiss
physician, Friedrich Miescher in 1869

5. FUNDAMENTAL STEPS OF DNA PURIFICATION
Sample Lysis
Removal contaminants from Nucleic acids
Concentration of Nucleic acids
Measurement of purity and concentration of Nucleic acids.

6. DNA purification: overview
cell growth cell harvest and lysis
DNA purification
DNA concentration

7. CELL LYSIS
Mechanical Method
Grinding in Liq N2
Sonication
Homogenization
Heat
Chemical method
SDS
Triton X-100
CTAB
Enzymatic Method
Lysozyme
Zymolase & Murienase
Proteinsae K

8. Separation of Nucleic acids from the contaminants
 Organic (Phenol-Chloroform) Extraction
Non-Organic (Proteinase K and Salting out)
Chelex (Ion Exchange Resin) Extraction
Silica Based
FTA Paper (Collection, Storage, and Isolation)

9. ORGANIC SOLVENT EXTRACTION METHOD

10. Adsorption Chromatography Method
Step 1: Prepare crude lysate
Silica-gel membrane
Apply to column
Step 2: Adsorb to silica surface
Centrifuge
Flow through
(discard)
Nucleic acids
Surface silanol groups are weakly
acidic, and will repel nucleic acids at
near neutral or high pH due to their
negative charge
Extraction Buffer composition favors
DNA and RNA adsorption to silica:
• Low pH
• High ionic strength
• Chaotropic salt Nucleic acids bind to the membrane,
while contaminants pass through the
column.

11. Adsorption Chromatography Method
Centrifuge
Nucleic acids
Step 3: Wash away residual contaminants
Wash buffer
Nucleic acids
Flow through
(discard)
Nucleic acids
Elution buffer
Elution Buffer composition is unfavorable to
surface binding:
High pH
Low ionic strength
Step 4: Elute nucleic acids
Centrifuge
Nucleic acids

12. FTATM Paper Extraction
 Cellulose based storage paper
 Extraction/storage of nucleid acids
from blood, buccal cells, tissue,
cultured cells, microorganisms,
plant tissue and other..
 DNA is stable at room temperature
for years

13. Fast and efficient DNA preparation: punch and purify
 Apply sample on FTA paper and let dry
 Cells are lysed on contact
 Use punch to add sample to tube
 Wash to remove PCR inhibitors
 Add „punch“ directly to PCR reaction.

14. FTA purification reagent (whatmann):
 For purification of nucleic acids stored on FTA
cards
 Ensures superior quality dna for pcr
 Removes heme, pcr inhibitors and other potential
contaminants. Non-toxic, hypoallergenic aqueous
solution.

15. FTA PAPER
 Exposes nucleic acids
- lyses cells and organelle membranes
- physically entraps nucleic acids
 Preserves and protects nucleic acids
- prevents damage by UV/ free radicals
- prevents enzymatic damage
- inhibits fungi and microbial growth
 Provides user safety
- inactivates potentially harmful viruses
http://www.dnasafestorage.com/httpdocs/FTA%20El
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16. CONCENTRATION OF THE GENOMIC DNA
70% final conc.
“spooling” Ethanol precipitation
Ethanol depletes the hydration shell surrounding
DNA and Reduces repulsive forces between
DNA strands which Causes aggregation and
precipitation of DNA

17. Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA plasmids
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate

18. Plasmid purification: alkaline lysis
Alkaline
conditions
denature DNA
Neutralize:
genomic DNA
can’t renature
(plasmids can
because they
never fully
separate)

19. Problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular transesterification
RNA is susceptible to nearly ubiquitous RNA-degrading
enzymes (RNases)
RNases are released upon cell lysis
RNases are present on the skin
RNases are very difficult to inactivate
-- disulfide bridges conferring stability
-- no requirement for divalent cations for activity

20. Common sources of RNase and how to avoid them
Contaminated solutions/buffers
 Use good sterile technique
 Treat solutions with depc (when possible)
 Make small batches of solutions
Contaminated equipment
 Use “rna-only” pipets, glassware, gel rigs
 Bake glassware, 300°c, 4 hours
 Use “rnase-free” pipet tips
 Treat equipment with depc

21. Top 10 sources of RNAse contamination
(Ambion Scientific website)
1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)
10) Enzyme preparations

22. Inhibitors of Rnase
DEPC:
 Diethylpyrocarbonate alkylating agent, modifying proteins and
nucleic acids fill glassware with 0.1% DEPC, let stand overnight
at room temp.
Solutions may be treated with depc -- add depc to 0.1%, then
autoclave (DEPC breaks down to CO2 and ethanol).

23. Making and using mRNA (1)

24. Making and using mRNA (2)

25. Purifying RNA: the key is speed
Break the cells/solubilize components/inactivate RNAses
by the addition of guanidinium thiocyanate (very powerful
denaturant)
Extract RNA using phenol/chloroform (at low pH)
Precipitate the RNA using ethanol/LiCl
Store RNA:
in DEPC-treated H20 (-80°C)
in formamide (deionized) at -20°C.

26. Selective capture of mRNA: oligo dT-cellulose
Oligo dT is linked to cellulose matrix
RNA is washed through matrix at high salt concentration
Non-polyadenylated RNAs are washed through
polyA RNA is removed under low-salt conditions
(not all of the non-polyadenylated RNA gets removed).

27. Other methods to capture mRNA
Poly(U) sepharose chromatography
Poly(U)-coated paper filters
Streptavidin beads:
•A biotinylated oligo dT is added to guanidinium-treated cells, and it
anneals to the polyA tail of mRNAs
•Biotin/streptavidin interactions permit isolation of the mRNA/oligo dT
complexes

28. NUCLEIC ACID ANALYSIS
 DNA or RNA is characterized using several different methods for
assessing quantity, quality, and molecular size.
◦ UV spectrophotometry
◦ Agarose gel electrophoresis
◦ Fluorometry
◦ Colorimetric blotting.

29. Quantity from UV Spectrophotometry
 DNA and RNA absorb maximally at 260 nm.
 Proteins absorb at 280 nm.
 [dsDNA] = (A260) X dilution factor X 50 µg/mL
 [ssDNA] = (A260) X dilution factor X 33 µg/mL
 [RNA] = (A260) X dilution factor X 40 µg/mL
 [Oligonucleotides] = (A260) X dilution factor X 20-
30µg/mL
Quantity from UV
Spectrophotometry

30. The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for
ssRNA. Ratios lower than 1.7 usually indicate significant
protein contamination.
The A260/A230 ratio of DNA and RNA should be roughly
equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower
ratios may indicate contamination by organic compounds
(e.g. phenol, alcohol, or carbohydrates).
Quality from UV Spectrophotometry

31. Quality from Agarose Gel Electrophoresis
 Genomic DNA:
◦ 0.6% to 1% gel, 0.125 µg/mL ethidium bromide in gel
and/or in running buffer
◦ Electrophorese at 70–80 volts, 45–90 minutes.
 Total RNA:
◦ 1% to 2% gel, 0.125 µg/ml ethidium bromide in gel
and/or in running buffer
◦ Electrophorese at 80–100 volts, 20–40 minutes.

32. 100 bp ladder
1 kb ladder
Lambda DNA cut
with Hind III
Lambda DNA
48,500 bp
(48.5 kb)
12,218 bp
23,130 bp
9,416 bp
6,557 bp
4,361 bp
2,322 bp
2,027 bp 517 bp
1,636 bp
3,054 bp
6,018 bp
100 bp
300 bp
600 bp
1,000 bp
1,500 bp
1,018 bp
2,036 bp
DNA Size from Agarose Gel Electrophoresis: Compares
unknown DNA to known size standards

33. Lambda DNA
marker
Human Whole Blood DNA
Lambda DNA cut with
Hind III marker
Whole blood genomic DNA
DNA Quality from Agarose Gel
Electrophoresis

34. Degraded RNA
DNA
28S
18S
5S rRNA, tRNA,
and other small
RNA molecules
mRNA = background smear
high  low MW
100 50 25 ng
Genomic DNA
markers
Cultured Cell RNA

35. Storage Conditions
 Store DNA in TE buffer at 4 °C for weeks or at –20 °C
to –80 °C for long term.
 Store RNA in RNase-free ultra pure water at –70 °C.