1. TYPES OF ISOLATION

2. 1. Organic Isolation Must purify DNA by removing contaminants.
an organic mixture of phenol and chloroform.
To avoid RNA contamination add RNAse, enzyme that degrades RNA.

3. 1. Organic Isolation Cont…..
Phenol/Chloroform
Biphasic
Hydrophobic layer on bottom has cell debris.
Hydrophilic layer on top has dissolved DNA
Remove top layer,
add cold ethanol,
DNA precipitates out.

4. 2. Inorganic Isolation Methods
Also called “salting out”.
Uses low pH and high salt condition to selectively precipitate proteins.
DNA is left in solution (picture on left).
2. Precipitate out DNA with isopropanol (right side picture). 2 1

5. 3.Solid Phase Isolation More rapid and effective
Use solid matrix to bind the DNA.
Wash away contaminants.
Elute DNA from column

6. Solid Phase Isolation cont…..
The diagram below explains the attractive properties of solid phase for DNA and RNA.
DNA/RNA hydrophilic……..Absorb
Lipids/protiens……. Hydrophobic……wash away

7. Isolation of Mitochondrial DNA
Mitochondrial DNA is passed from generation to generation along the maternal lineage.
Centrifugation to separate out
Lyse
Precipitate with cold ethanol.

8. Nuclear DNA Present in almost every cell
Combination from both parents; 23 chromosomes from each parent

9. Mitochondrial DNA
Each cell contains thousands of mt, each containing copies of its DNA
Mt DNA is in larger quantities in a cell
Nuclear DNA is larger in size

10. Mt DNA is 16,569 bases in length and consists of 2 different regions
Coding Region
Produces
13 proteins,
22 tRNAs
2 rRNAs
This region has very little variability
So everyone’s DNA in this region will be nearly the same sequence of

11. Human Mitochondrial Genome
Human mtDNA composes of a control region (CR)

12. 610 bp with a lot of variability
Control Region
This region is highly variable within the human population
Consists of 2 subregions
HV1 = 342 bp HV2 = 268
610 bp with a lot of variability

13. Mutations occur in the control region of mt

14. How is mtDNA isolated? Compare your sequence …… prehistoric DNA
Isolate DNA from cheek cells
Complete a PCR reaction
Produce millions of extra copies of HV1 on the control region of mtDNA
Send amplified DNA away to be sequenced (Identify the exact sequence of HV1 in your mtDNA)
Compare your sequence …… prehistoric DNA

16. Isolation of RNA
Requires STRICT precautions to avoid sample degradation.

17. RNAses RNases are naturally occurring enzymes that degrade RNA
Common laboratory contaminant (from bacterial and human sources)
Also released from cellular compartments during isolation of RNA from biological samples
Can be difficult to inactivate

18. RNAses cont…… MUST be eliminated or inactivated BEFORE isolation.
RNAses are enzymes which are small proteins that can renature and become active.
MUST be eliminated or inactivated BEFORE isolation.
CRITICAL to have a separate RNAse free area of lab.

19. Protecting Against RNAse
Wear gloves at all times
Use RNase-free tubes and pipet tips
Use dedicated, RNase-free, chemicals
Pre-treat materials with extended
heat (180 C for several hours),
wash with DEPC-treated water (Di ethyl pyro carbonate)
Supplement reactions with RNase inhibitors

20. Total RNA 80-90% of total RNA is ribosomal RNA.
2.5-5% is messenger RNA

21. Organic RNA Extraction
Lyse/homogenize cells
Add phenol:chloroform:isoamyl alcohol to lysed sample, and centrifuge
Organic phase separates from aqueous phase
Organic solvents on bottom
Aqueous phase on top (contains total RNA)
Remove RNA solution to a clean tube;
precipitate RNA and
wash with ethanol, then
resuspend RNA in water

22. Types of nucleic acid Viral RNA Bacterial RNA Genomic RNA Viral DNA
Bacterial DNA
Genomic DNA
Plasmid
mitochondrial

24. Chloroform causes TRI Reagent
combines phenol and guanidine thiocyanate in a mono-phase solution to facilitate the immediate and most effective inhibition of RNase activity.
A biological sample is homogenized or lysed in TRI Reagent .
guanidinium isothiocyanate 
powerful protein denaturant)
RNA is stable in trizol which deactivates RNases.
Chloroform causes
proteins to become denatured and
become soluble in the organic phase or interphase,
while nucleic acids remain in the aqueous phase.

25. Isopropanol
RNA is
insoluble in isopropanol so it will aggregate together, giving a pellet upon centrifugation.
This step also removes alcohol-soluble salt.