Relevance of DNA Isolation Isolation of DNA is often the first

step before further analysis

• DNA profiling

• Cloning

• Disease diagnosis

• DNA sequencing

• Genetically modified organisms (GMO) -agriculture, pharmaceutical

• Environmental testing, biodefense

1. Cell Collection

Gently chewing the inside of the mouth combined with a water mouth wash is used to dislodge epithelial cells lining the mouth

Ample cell collection is critical for success.

2. Lysis Buffer

What is Lysis Buffer?

• 50 mM Tris-HCI, pH 8.0• 1% SDS

Tris buffer to maintain the pH of the solution at a level where DNA is stable

1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage).

O

S

O

O

O

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

SDSNa+

3. Why Add Protease?

•Protease is added to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA.

•Protease treatment increases the amount of intact DNA that is extracted.

4. Adding Salt

• The protease solution already contains salt

• Na+ ions of NaCI bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules.

• The addition of NaCI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added.

Na+

Na+

Na+

Na+

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O

DNA Structure

5. Adding Ice Cold Alcohol?

•DNA does not dissolve in alcohol.

•The addition of cold alcohol makes the DNA clump together and precipitate out of solution.

•Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands.

•Microscopic oxygen bubbles “aggregate” , or “fuse” together, as the DNA precipitates.

•The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase.

DNA Fingerprinting Real WorldApplications • Crime scene

• Human relatedness

• Paternity

• Animal relatedness

Anthropology studies

Disease-causing organisms

Food identification

Human remains

Monitoring transplants

DNA Restriction Enzymes

• Evolved by bacteria to protect against viral DNA infection

• Endonucleases = cleave within DNA strands

• Over 3,000 known enzymes

Enzyme Site Recognition

• Each enzyme digests (cuts) DNA at a specific sequence = restriction site

• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)

Palindrome

Restriction site

Fragment 1 Fragment 2

5 vs 3 Prime Overhang

• Generates 5 prime overhang

Enzyme cuts

Common Restriction Enzymes

EcoRI– Eschericha coli– 5 prime overhang

Pstl– Providencia stuartii– 3 prime overhang

The DNA DigestionReaction

Restriction Buffer provides optimal conditions

• NaCI provides the correct ionic strength

• Tris-HCI provides the proper pH

• Mg2+ is an enzyme co-factor

DNA DigestionTemperature

Why incubate at 37°C?

• Body temperature is optimal for these and most other enzymes

What happens if the temperature is too hot or cool?

• Too hot = enzyme may be denatured (killed)

• Too cool = enzyme activity lowered, requiring longer digestion time

Restriction Fragment Length PolymorphismRFLP

Allele 1

Allele 2

GAATTCGTTAAC

GAATTCGTTAAC

CTGCAGGAGCTC

CGGCAGGCGCTC

PstI EcoRI

1 2 3

3Fragment 1+2Different Base PairsNo restriction site

+

M A-1 A-2

Electrophoresis of restriction fragments

M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments

AgaroseElectrophoresisLoading

• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode

Power Supply

Buffer

Dyes

Agarose gel

AgaroseElectrophoresisRunning

• Agarose gel sieves DNA fragments according to size– Small fragments

move farther than large fragments

Power Supply

Gel running

Analysis of Stained Gel

Determinerestriction fragmentsizes

• Create standard curve using DNA marker

• Measure distance traveled by restriction fragments

• Determine size of DNA fragments

Identify the relatedsamples

Molecular Weight Determination

Size (bp) Distance (mm)

23,000 11.0 9,400 13.0

6,500 15.0

4,400 18.0

2,300 23.0

2,000 24.0 100

1,000

10,000

100,000

0 5 10 15 20 25 30

Distance, mm

Siz

e, b

ase

pai

rsB

A

Fingerprinting Standard Curve: Semi-log