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Extraction of Human DNA

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Title: Extraction of Human DNA


1
Extraction of Human DNA
2
Experiment Goals
  • Isolation of genomic DNA from human blood
  • Analysis of isolated DNA using
  • Agarose gel electrophoresis
  • Spectrophotometry

3
What is a DNA?
  • DNA, also known as deoxyribonucleic acid,
  • A fundamental molecule found in all living things
  • Carries the genetic information in the cell
  • Contains instructions for our body cells to
    perform their specific functions
  • The sequence of nucleotides determines individual
    hereditary characteristics

4
What is a DNA?
  • Basic unit of information in DNA is the gene
  • Human beings have about 30,000 gene
  • Size of organisms genome is roughly a measure of
    its complexity
  • Viruses 5-10 kb
  • E. coli 4,640 kb
  • Human 2,900,000 kb

5
DNA Extraction
  • DNA extraction is a routine procedure to isolate
    collect DNA.
  • DNA extraction is the first step for subsequent
    molecular or forensic analysis.
  • DNA can be extracted from almost any intact
    cellular tissue
  • Skin,
  • blood,
  • saliva,
  • semen,
  • mucus,
  • muscle tissue,
  • bone marrow, etc.

6
Nucleic Acid Preparation Applications
  • Medical studies
  • Understanding genetic disorders at molecular
    level
  • Rapid detection of genetic disorders in a patient
  • Agricultural studies
  • Plant and animal breeding
  • Criminology/Paternity testing
  • DNA fingerprinting to identify individuals.

7
Basic steps in DNA extraction
  • There are three basic steps in a DNA extraction,
    the details of which may vary depending on the
    type of sample and any substances that may
    interfere with the extraction and subsequent
    analysis.
  • Break open cells and remove membrane lipids
  • Remove cellular and histone proteins bound to the
    DNA, by adding a protease, by precipitation with
    sodium or ammonium acetate, or by using a
    phenol/chloroform extraction step.
  • Precipitate DNA in cold ethanol or isopropanol,
    DNA is insoluble in alcohol and clings together,
    this step also removes salts.

8
Procedure
9
Overview of Procedure
10
Blood Collection
  • Blood collected in disodium EDTA tube
  • Samples can be stored at -20oC or -70oC
  • Fresh samples are kept in freezer for a few hours
    to facilitate RBCs hemolysis
  • Allow samples to thaw before starting the
    extraction

11
1- RBCs Lysis
  • Pipette 3 mls of whole blood in a conical
    centrifuge tube
  • Add 9 mls of 1X erythrocyte lysing buffer (0.155M
    NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4)
  • Leave 10 min. at RT, mix occasionally
  • Centrifuge at 4000 rpm for 5 min
  • Discard supernatent
  • White pellet is observed at bottom of tube
  • Wash pellet 3 times by adding 3 mls of buffer,
    incubate 10 min at RT, centrifuge

12
2- WBCs nuclei Lysis proteins digestion
  • Add 1.5 mls of SE buffer (75mM NaCl, 25 mM
    Na2EDTA, pH 8.0) containing 100µg/ml of
    Proteinase K 1 sodium dodecyl sulphate (SDS)
    to the pellet
  • Incubate at 37-55oC overnight in a water bath or
    incubator
  • WBCs nuclei denatured DNA goes out in solution

13
3- Separate contaminants from DNA
  • After incubation add 1.5 mls of SE buffer, 750 µl
    of 6M NaCl 3.75 mls chloroform
  • Mix vigorously on vortex for 20 sec
  • Mix for 30 min (on rotator)
  • Centrifuge for 10 min at 2000 rpm
  • 2 phases are observed
  • DNA is extracted in supernatant proteins in the
    lower phase
  • Transfer upper aqueous phase (containing DNA) to
    a clean tube

14
4- Precipitate DNA
  • Add an equal volume of isopropanol
  • DNA will be precipitated by gentle swirling
    observed as a white thread like strand
  • Using a sterile spatula or loop transfer the DNA
    strand into a sterile microcentrifuge tube
    containing 1 ml of 75 ethanol
  • Wash by inversion to remove any remaining salts
  • Centrifuge, discard supernatent
  • Repeat the washing step, then centrifuge
  • Remove supernatant, and dry the pellet

15
5- Resuspend DNA in final buffer
  • Dried pellet is resuspended in TE buffer and left
    overnight on a rotator

16
DNA Analysis
  • Different methods for assessing quantity
    quality of extracted DNA
  • Agarose gel electrophoresis
  • UV spectrophotometry

17
Checking the Quality of DNA
  • The product of DNA extracted will be used in
    subsequent experiments
  • Poor quality DNA will not perform well in PCR

18
Quality from Agarose Gel Electrophoresis
  • Quality of DNA extracted is assessed using the
    following simple protocol
  • Mix 5 µL of DNA with 5 µL of loading Dye
  • Load this mixture into a 1 agarose gel
  • Stain with ethidium bromide
  • Electrophorese at 7080 volts, 4590 minutes.

19
DNA Quality fromAgarose Gel Electrophoresis
  • High molecular weight band
  • Smearing indicates DNA degradation

20
Nucleic Acid Characterization
  • Absorption Spectra
  • Absorb light in ultraviolet range, most strongly
    in the 254-260 nm range
  • Useful for quantification of samples

21
Quantity from UV Spectrophotometry Calculating
Yield
22
Spectrophotometric analysis of DNA
23
Quality from UV Spectrophotometry
  • DNA absorb maximally at 260 nm.
  • Proteins absorb at 280 nm.
  • Background scatter absorbs at 320 nm.

24
Quality from UV Spectrophotometry
25
Storage Conditions
  • Store DNA in TE buffer at 4 C for weeks or at
    20 C to 80 C for long term.
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