DNA Extraction

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DNA Extraction

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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What is DNA?

• What does DNA stand for?

Deoxyribose Nucleic Acid or Deoxyribonucleic
Acid
What does DNA do?

• DNA contains genetic information.
• DNA codes for the proteins our bodies make that
  are necessary for survival.

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What is DNA?

• DNA is a code for making proteins

AGC TAG CTT ATA CTC TAT CTC TTT
Amino Acid
Amino Acid
Amino Acid
Amino Acid
Amino Acid
Amino Acid
The order of amino acids determines what type of
protein is made.
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What is DNA?

• Some common proteins are

• Hemoglobin - carries oxygen from lungs to cells
• Insulin - regulates metabolism
• Many types of enzymes - catalyze reactions in the
  body, such as the breakdown of sugar for energy

DNA also determines how much of these proteins
each cell makes.
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What is DNA?

• What does DNA look like?

Double Helix
Like a Twisted Ladder
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What is DNA?

• What does DNA look like?

Sugar Phosphate Backbone (Sides of Ladder)
Nitrogenous Base (Rungs of Ladder)
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What is DNA?

• The DNA ladder is made up of building blocks
  called nucleotides.

What is a nucleotide?
Adenine Cytosine Guanine Thymine
Phosphate Group
Base
Deoxyribose sugar
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The 4 Bases
A Adenine
C Cytosine
T Thymine
G Guanine
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The 4 Bases
A
C
T
G
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The 4 Bases
The bases pair up to form the rungs of the ladder.
A pairs with T
G pairs with C
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What is DNA?

• DNA is written as the sequence of these bases

AAGTCGATCGATCATCGATCATACGT

• Only one side of the ladder is written.

• In humans, there are three billion base pairs
  (letters) in the DNA within each cell.

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What is DNA?

• Polynucleotides can be any length and have any
  sequence

• Several thousand basepairs length in lab
• Millions in chromosomes

No chemical restrictions on order

• 10 in length 410 1,048,576 sequences
• Variability enables genetic material to exist in
  an infinite number of forms

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What is DNA?

• Among humans, most of the 3 billion bases in the
  DNA sequence are exactly the same.

• Our Human DNA is 99.8 similar to each other, but
  the 0.2 difference is more than enough to
  distinguish us from one another.

• Human DNA is even 98 similar to chimpanzees.

• NO TWO PEOPLE HAVE IDENTICAL DNA
• except identical twins

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Where is DNA?

• DNA is found in the cells in our body.

Nucleus (Brain of the cell)
Mitochondria (more later)
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Where is DNA?

• All types of cells in our body contain a copy of
  the same DNA.

Some cells important to forensic science are
White Blood Cell
Sperm Cell
Cheek Cell
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Where is DNA?

• DNA in the nucleus is packaged into chromosomes

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Where is DNA?
Chromosomes come in pairs
(one from Mother) (one from Father)
There are 46 chromosomes in each cell.
(23 pairs)
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Where is DNA?

• What are sources of DNA at a crime scene?

DNA can be recovered from any substance that
contains cells.

• Blood
• Semen
• Saliva
• Tissue

• Bone
• Teeth
• Hair
• Maggot Crops

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Evidence Collection
Blood and semen stains are the two most commonly
tested biological evidence.

• Moisture allows for bacterial growth and destroys
  DNA. Therefore
• Stains should be thoroughly air dried.
• Samples should be packaged in paper envelopes,
  not plastic bags.

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Evidence Collection
When stains on immovable objects cannot be
transported back to the lab, they can be swabbed.

• Swab moistened with distilled water.
• Air dried before packaging.
• All swabs in separate envelopes.
• Swab near area for negative control.

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Evidence Collection
Other Tips

• No contamination. Do not touch area with bare
  hands, sneeze or cough over evidence.
• Clean latex gloves for collecting each item.
• Each item of evidence packaged separately.

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Evidence Collection
Evidence Storage

• Best bet is dry and cold. This reduces rate of
  bacterial growth and DNA degradation.
• Store at 4ºC (refrigeration), -20ºC (freezer),
  -70ºC (deep freezer).

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Extraction

• From Forensic Samples

• Cells broken open to release contents
• Cell extract treated to remove all components
  except DNA
• DNA solution is concentrated

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Extraction

• Preparing Cell Extract

• Initial methods vary based on the type of sample
  being processed.

• All methods involve breaking open cells and
  getting cellular material into a solution.

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Extraction

• Preparing Cell Extract

Liquid sample cells already in solution

• Stain/swab/other
• Material soaked and cells are released into
  solution. Physical agitation may assist.
• Loose cells may be concentrated by centrifuging
  to pellet, and then removing supernatent (before
  cell lysis).

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Extraction
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Extraction

• Preparing Cell Extract

Cell must be broken open to release DNA

• Physical Methods
• Mechanical force breaks cell membrane/wall
• Mortar and pestle sonication
• Not usually used for DNA prep

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Extraction

• Preparing Cell Extract

Cell must be broken open to release DNA

• Chemical Methods
• Attack cell wall and cell membrane
• Cell wall lysozyme, EDTA or both
• Cell membrane detergent (SDS)

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Extraction

• Preparing Cell Extract

• EDTA binds Mg ions
• inhibits enzymes that can degrade DNA
• helps break down cell wall

• Cell membrane disruption
• Sodium dodecyl sulphate (SDS) is a detergent
  removes lipid molecules

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Extraction
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Extraction

• Preparing Cell Extract

Some material may require special treatment
Fingernails, hair, sperm cells require use of DTT
(dithiothreitol) to break down membrane and
extracellular material.
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Extraction
Centrifuge removes insoluble cell debris
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Extraction

• Purification of DNA

• In addition to DNA, a large amount of proteins
  and RNA remain.
• These must be removed to avoid interference with
  further analysis

Several way to remove RNA and proteins
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Chelex Extraction

• Chelex 100
• Ion-exchange resin small beads
• Binds metal ions (magnesium)
• The removal of magnesium inactivates DNA
  destroying enzymes.
• DNA left in supernatant

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Chelex Extraction
Chelex procedure

• Initial wash to remove proteins or other
  inhibitors. (proteinase K)
• Samples are boiled
• Breaks open cells
• Destroys cell proteins
• Also, denatures DNA
• Chelex spun to bottom DNA in solution

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Chelex Extraction
Chelex Traits

• Quick, cheap, and easy
• Single stranded DNA suitable for PCR
• Not as sensitive (for low-level DNA)
• RNA also in solution

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Organic Extraction

• Purification of DNA (Organic Extraction)

• Add phenol or phenol/chloroform (11).
• Proteins are precipitated form white layer at
  interface between organic and aqueous layers.
• Aqueous solution removed.

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Organic Extraction

• Purification of DNA

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Organic Extraction

• Purification of DNA

What if there are excessive proteins?

• Repeated phenol extractions
• Not favorable DNA destruction with mixing

• Breakdown with protease before extraction
• Pronase or proteinase K

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Organic Extraction

• Purification of DNA

• Some mRNA removed with phenol treatment.
• Remaining RNA can be digested with ribonuclease
  (if necessary)

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Organic Extraction

• Concentration of DNA

• If a small amount of DNA is targeted, the
  resulting solution will be dilute.

• One method is precipitation by ethanol.
  Precipitate centrifuged supernatent removed.

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Organic Extraction

• Concentration of DNA

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Silica Extraction

• Purification of DNA

• Guanidinium thiocyanate added to extract
• Denatures non-DNA material
• Makes DNA bind to silica particles
• Silica added directly or sample passed through
  silica column.
• DNA removed by adding water

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Silica Extraction

• DNA purification with silica particles

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Special Extractions

• Differential Extraction

In rape kit swab, both female epithelial and male
sperm cells will be found

• First, female cells are broken open with
  SDS/proteinase K.
• Sperm cells are collected through centrifugation.
• Sperm cells are digested using SDS/proteinase
  K/DTT

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Special Extractions

• Plant cells/Bacteria cells

• Cell wall disruption
• Lysozyme digests polysaccharides structural
  components of cell wall
• EDTA (ethylenediamine tetraacetate) binds
  magnesium ions essential for preserving structure
  of cell and inhibits enzymes that could degrade
  DNA

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Special Extractions

• Plant Cells

• Plant cells have high carbohydrate content
• Lysozyme has no effect
• Cetyltrimethylammonium (CTAB) added binds
  nucleic acid and precipitates
• Centrifuged supernatant removed

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Special Extractions

• Purification of Plant DNA