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Relevance of DNA Isolation PowerPoint PPT Presentation


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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.

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Relevance of DNA Isolation

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Relevance of dna isolation

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


Cell bio 101 what are the structures of the cell

Cell Bio 101: What are the Structures of the Cell?


Relevance of dna isolation

Cell Structures


Protocol highlights genomic dna extraction

Protocol Highlights: Genomic DNA Extraction

  • •Use a simple water mouthwash to collect cheek cells

  • •Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents

  • •Digest sample with protease to degrade proteins

  • •Precipitate DNA with cold alcohol in high salt


Genes in a bottle kit

Genes in a Bottle Kit

  • Why Perform Each Step?


Relevance of dna isolation

Cell CollectionGently 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.


Relevance of dna isolation

CH3

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

O

O

O

S

-

O

SDS

2. Lysis BufferWhat is Lysis Buffer?• 50 mM Tris-HCI, pH 8.0• 1% SDSTris buffer to maintain the pH of the solution at a level where DNA is stable1% 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).

Na+


3 why add protease

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

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.


Relevance of dna isolation

O

Na+

O

P

O

Base

Na+

O

O

CH2

Sugar

O

Na+

O

O

P

Base

Na+

O

O

CH2

Sugar

OH

DNA Structure


5 adding ice cold alcohol

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 world applications

DNA Fingerprinting Real WorldApplications

  • •Crime scene

  • •Human relatedness

  • •Paternity

  • •Animal relatedness

  • Anthropology studies

  • Disease-causing organisms

  • Food identification

  • Human remains

  • Monitoring transplants


Model of dna dna is comprised of four base pairs

Model of DNADNA is Comprised of Four Base Pairs


Deoxyribonucleic acid dna

Deoxyribonucleic Acid (DNA)


Dna schematic

DNASchematic

O

Phosphate

O

P

O

Base

O

O

CH2

Sugar

O

O

O

P

Phosphate

Base

O

O

CH2

Sugar

OH


Dna restriction enzymes

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

Enzyme Site Recognition

Restriction site

Palindrome

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

•Enzymes recognize 4- or 6- base pair, palindromic sequences

(eg GAATTC)

Fragment 2

Fragment 1


5 vs 3 prime overhang

5 vs 3 Prime Overhang

Enzyme cuts

•Generates 5 prime overhang


Common restriction enzymes

Common Restriction Enzymes

EcoRI

– Eschericha coli

– 5 prime overhang

Pstl

– Providencia stuartii

– 3 prime overhang


The dna digestion reaction

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 digestion temperature

DNA DigestionTemperature

  • Why incubate at 37°C?

  • •Body temperature is optimal for these and most other enzymes

  • What happens if the temperature istoo hot or cool?

  • •Too hot = enzyme may be denatured (killed)

  • •Too cool = enzyme activity lowered, requiring

  • longer digestion time


Restriction fragment length polymorphism rflp

PstI

EcoRI

Restriction Fragment Length PolymorphismRFLP

GAATTC

GTTAAC

CTGCAG

GAGCTC

Allele 1

1

2

3

CGGCAG

GCGCTC

GAATTC

GTTAAC

Allele 2

3

Fragment 1+2

Different

Base Pairs

No restriction site

M

A-1

A-2

Electrophoresis of restriction fragments

M: Marker

A-1: Allele 1 Fragments

A-2: Allele 2 Fragments

+


Agarose electrophoresis loading

AgaroseElectrophoresisLoading

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

Buffer

Dyes

Agarose gel

Power Supply


Agarose electrophoresis running

AgaroseElectrophoresisRunning

  • •Agarose gel sieves DNA fragments according to size

  • –Small fragments move farther than large fragments

Gel running

Power Supply


Analysis of stained gel

Analysis of Stained Gel

  • Determine

  • restriction fragment

  • sizes

  • •Create standard curve using DNA marker

  • •Measure distance traveled by restriction fragments

  • •Determine size of DNA fragments

  • Identify the related

  • samples


Molecular weight determination

Molecular Weight Determination

Fingerprinting Standard Curve: Semi-log

  • Size (bp)Distance (mm)

  • 23,00011.0

  • 9,40013.0

  • 6,50015.0

  • 4,40018.0

  • 2,30023.0

  • 2,00024.0


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