Extraction of Human DNA

Extraction of Human DNA

Experiment Goals

• Isolation of genomic DNA from human blood

• Analysis of isolated DNA using– Agarose gel electrophoresis– Spectrophotometry

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

What is a DNA?

• Basic unit of information in DNA is the gene

• Human beings have about 30,000 gene

• Size of organism’s genome is roughly a measure of its complexity

• Viruses 5-10 kb

• E. coli4,640 kb

• Human 2,900,000 kb

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.

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.

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.

Procedure

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 Procedure

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

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

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

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

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

5- Resuspend DNA in final buffer

• Dried pellet is resuspended in TE buffer and left overnight on a rotator

DNA Analysis

• Different methods for assessing quantity & quality of extracted DNA– Agarose gel electrophoresis– UV spectrophotometry

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

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 70–80 volts, 45–90 minutes.

DNA Quality fromAgarose Gel Electrophoresis

• High molecular weight band

• Smearing indicates DNA degradation

Nucleic Acid Characterization

• Absorption Spectra– Absorb light in ultraviolet range, most strongly

in the 254-260 nm range• Useful for quantification of samples

Multiply the concentration of theDNA sample by the

volume of hydrating solution added.

Example for DNA: 150 µg/mL X 0.1 mL = 15 µg

Concentration from UV Spec. (µg DNA per ml of hydrating solution)

Volume of hydration solution

DNA yield

Quantity from UV Spectrophotometry Calculating Yield

Spectrophotometric analysis of DNA

Purity 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 orother contaminant

Purity from UV Spectrophotometry

Storage 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

Recommendedfor long-term

storage in ethanol

<4 Months 1–3 Years <7 Years >7 Years