Extraction of Human DNA
Experiment GoalsExperiment Goals
•Isolation of genomic DNA from humanblood.
•Analysis of isolated DNA using
Agarose gel electrophoresis
Spectrophotometry
What is a DNA?What is a DNA?
•DNA, also known as deoxyribonucleicacid.
•A fundamental molecule found in all livingthings.
•Carries the genetic information in the cell.
•Capable of self-replication and synthesisof RNA.
•Contains instructions for our body cells toperform their specific functions.
•RNA. DNA consists of two long chains ofnucleotides twisted into a double helix
•Joined by hydrogen bonds between thecomplementary bases adenine and thymine orcytosine and guanine.
•The sequence of nucleotides determinesindividual hereditary characteristics.
DNA plays an important role in two processes
•The process of replication, DNA providesinformation to copy itself, so genetic informationcan be passed on from generation to generationof cells.
•also provides instructions for making proteins,which are vital to the maintenance and functionof cells.
What is a DNA?What is a DNA?
•Basic unit of information inDNA is the gene
•Human beings have about30,000 gene
•Size of organism’s genome isroughly a measure of itscomplexity
•Viruses5-10 kb
•E. coli4,640 kb
•Human2,900,000 kb
• (about 2.9 billion base pairs)
•Only about 5% code for protein. Interveningsequences and other noncoding sequences makeup the remainder.
•In addition to genomic DNA, mitochondria, whichare cellular organelles, contain their own DNA(mitochondrial DNA) which replicateindependently from cell chromosomal DNA.
DNA ExtractionDNA Extraction
•DNA extraction is a routine procedure to isolate & collectDNA.
•DNA extraction is the first step for subsequent molecularor forensic analysis.
•DNA can be extracted from almost any intact cellulartissue
•Skin,
•blood,
•saliva,
•semen,
•mucus,
•muscle tissue,
•bone marrow, etc.
Nucleic Acid Preparation ApplicationsNucleic Acid Preparation Applications
•Medical studies
Understanding genetic disorders at molecularlevel.
Rapid detection of genetic disorders in apatient.
•Agricultural studies
Plant and animal breeding
•Criminology/Paternity testing
DNA fingerprinting to identify individuals.
Basic steps in DNA extractionBasic steps in DNA extraction
•There are three basic steps in a DNA extraction,the details of which may vary depending on thetype of sample and any substances that mayinterfere with the extraction and subsequentanalysis.
Break open cells and remove membrane lipids
Remove cellular and histone proteins bound to theDNA, by adding a protease, by precipitation withsodium or ammonium acetate, or by using aphenol/chloroform extraction step.
Precipitate DNA in cold ethanol or isopropanol, DNA isinsoluble in alcohol and clings together, this step alsoremoves salts.
1- Lyse RBCs & WBCs
2- Lyse WBCs nuclei & Denature/digest proteins
3- Separate contaminants (e.g., proteins, heme)
4- Precipitate DNA
5- Resuspend DNA in final buffer
Overview of ProcedureOverview of Procedure
Blood CollectionBlood Collection
•Blood collected in disodium EDTA tube.
•Samples can be stored at -20oC or -70oC.
•Fresh samples are kept in freezer for a fewhours to facilitate RBCs hemolysis.
•Allow samples to thaw before starting theextraction.
1- RBCs Lysis1- RBCs Lysis
•Pipette 3 mL of whole blood in a conicalcentrifuge tube.
•Add 9 mL of 1X erythrocyte lysing buffer.
•Leave 10 min. at RT, mix occasionally.
•Centrifuge at 4000 rpm for 5 min.
•Discard supernatant.
•White pellet is observed at bottom of tube.
•Wash pellet 3 times by adding 3 mL of buffer,incubate 10 min at RT, & centrifuge
2- WBCs nuclei Lysis & proteins digestion2- WBCs nuclei Lysis & proteins digestion
•Add 1.5 mL of SE buffer to the pellet
•Incubate at 37-55oC overnight in a waterbath or incubator
•WBCs nuclei denatured & DNA goes outin solution
3- Separate contaminants from DNA3- Separate contaminants from DNA
•After incubation add 1.5 mL of SE buffer, 750 µlof 6M NaCL & 3.75 mL 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 thelower phase.
•Transfer upper aqueous phase (containing DNA)to a clean tube.
4- Precipitate DNA4- 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 DNAstrand into a sterile micro centrifuge tubecontaining 1 mL of 75% ethanol.
•Wash by inversion to remove any remaining salts.
•Centrifuge at 11000 g for 4 minutes and thendiscard supernatant.
•Repeat the washing step, then centrifuge.
•Remove supernatant, and dry the pellet.
5- Resuspend DNA in final buffer5- Resuspend DNA in final buffer
•Dried pellet is resuspended in TE bufferand left overnight on a rotator
DNA AnalysisDNA Analysis
Different methods for assessing quantity & qualityof extracted DNA
Agarose gel electrophoresis
UV spectrophotometry
Checking the Quality of DNAChecking the Quality of DNA
•The product of DNA extracted will be usedin subsequent experiments
•Poor quality DNA will not perform well inPCR
Quality from Agarose Gel ElectrophoresisQuality from Agarose Gel Electrophoresis
•Quality of DNA extracted is assessedusing 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
•Electrophoreses at 70–80 volts, 45–90minutes.
•view on UV transilluminator.
DNA Quality fromAgarose Gel ElectrophoresisDNA Quality fromAgarose Gel Electrophoresis
•High molecularweight band
•Smearing indicatesDNA degradation
Nucleic Acid Characterization
•Absorption Spectra
–Absorb light in ultraviolet range, most stronglyin the 254-260 nm range
•Useful for quantification of samples
Multiply the concentration of the
DNA sample by the
volume of hydrating solution added.
Example for DNA: 150 µg/mL X 0.1 mL = 15 µg
Concentration from UVSpec. (µg DNA per mlof hydrating solution)
Volume ofhydrationsolution
DNA yield
Quantity from UV SpectrophotometryCalculating YieldQuantity from UV SpectrophotometryCalculating Yield
Spectrophotometric analysis of DNASpectrophotometric analysis of DNA
Quality from UV SpectrophotometryQuality from UV Spectrophotometry
•DNA absorb maximally at 260 nm.
•Proteins absorb at 280 nm.
•Background scatter absorbs at 320 nm.
A260/A280 = measure of purity
(A260 – A320)/(A280 – A320)
1.7 – 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant
Quality from UV SpectrophotometryQuality from UV Spectrophotometry
Storage ConditionsStorage Conditions
•Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term.
2–25 °C
2–8 °C
–20 °C
–70 °C
Recommended
for long-term
storage in ethanol
<4 Months
1–3 Years
<7 Years
>7 Years