DNA Estimation

Academic Script

INTRODUCTION

Dear friends, welcome! Today we are going to talk about how to estimate the
content of DNA from the tissue. Deoxyribonucleic acid (DNA) is the genetic blue print of
life found in all living organisms. It supplies all information from the parents necessary
for cells to reproduce through instruction of genes contained in it. It is also responsible
for determining how a person looks. DNA often contains mutated codes for diseases
that are genetic, passed from parents to child. DNA is also important for researchers
who determine the role of genes played in complex diseases such as cancer, diabetes
and heart disease.
A. HISTORY OF DNA
DNA was first discovered in 1869 by J. F.Miescher. Miescher identified a
substance found in the nucleus of white blood cells. This substance would later come to
be known as DNA. In 1912, studies done using new microscope technology showed
that both DNA and proteins are both present in chromosomes. Thirty-seven years later,
a biochemist named Erwin Chargaff proved that DNA is species specific. In 1953,
James Watson and Francis Crick were able to demonstrate the molecular structure of
DNA. These two scientists went on to win a Noble Prize for their discovery.

B. IDENTIFICATION
DNA is often referred to as a double helix because of its appearance. It is made
of two long strands called nucleotides that run in opposite directions from one another.
Nucleotides are made of sugar and phosphate groups that are joined together by ester
bonds. Attached to sugars is a molecule called a base. Four different types of bases
encode the information that is used for cell replication.

C. EVOLUTION
As organisms evolve, DNA sequences change to produce new qualities and
weed out qualities that are no longer needed. Sometimes this happens because of the
process of natural selection. Qualities that help people survive certain diseases and
conditions continue to be passed on to offspring; less desirable qualities are slowly
removed from the population. The DNA evolutions help species to survive and
reproduce despite changing conditions.
 D. INFORMATION
The information in DNA is stored as a code made up of four chemical bases i.e.
adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about
3 billion bases, and more than 99 percent of those bases are same in all people. The
order, or sequence, of these bases determine the information available for building and
maintaining an organism, similar to the way in which letters of the alphabets appear in a
certain order to form words and sentences.
DNA bases pair up with each other, A with T and C with G, to form units called
base pairs. Each base is also attached to a sugar molecule and a phosphate molecule.
Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are
arranged in two long strands that form a spiral called a double helix.
An important property of DNA is that it can replicate, or make copies of itself.
Each strand of DNA in the double helix can serve as a pattern for duplicating the
sequence of bases. This is critical when cells divide because each new cell needs to
have an exact copy of the DNA present in the old cell.
The bases present in DNA are adenine, guanine, cytosine and
thymine. Hydrogen bonds between the base portions of the nucleotides hold the two
chains together. The bases of one strand of DNA are paired with the bases of the
second strand, so that an adenine is always paired with a thymine and a cytosine is
always paired with a guanine.
Such base pairing results in one DNA strand being complementary to the other.
Hydrogen bonding between the DNA bases can consist of two associated
polynucleotide strands that wind together to form a double helix. The two sugar
phosphate backbones are on the outside of the double helix, and the bases project into
the interior. The orientation of the two strands is anti-parallel; that is, their 5´to
3´directionsare opposite.
The structure of the DNA is double helix showing hydrogen bonding between the
bases. DNA is present not only in chromosomes in the nucleus of eukaryotic organisms,
but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack
nuclei, have a single chromosome, but may also contain DNA in the form of plasmids.
DNA can be chemically identified and measured by the diphenylamine test.
Diphenylamine reagent is prepared using acetic acid and sulphuric acid. The structure
of diphenylamine is shown in contrast to the stability of the links between pyrimidines
and deoxyribose, those between purines and deoxyribose are very labile, so it appears
that diphenylamine reacts with the sugar residues originally combined with purines in
the DNA (Burton 1955).Under extreme acidic conditions, DNA becomes depurinated.
 Depurination is an alteration of DNA in which the purine base is removed from
the deoxyribosesugar by hydrolysis of the beta-N-glycosidic link between them. After
depurination, the sugar phosphate backbone remains and the sugar ring has a hydroxyl
(-OH) group in the place of the purine. Depurination of the DNA is followed by the
dehydration of sugar to hydroxylevulinylaldehyde which in turn reacts with
diphenylamine to give blue colour. The intensity of the blue color is proportional to the
concentration of DNA in the sample and this could be measured via spectrophotometry.

E. SIGNIFICANCE
DNA is the building block of all organisms. Looking for mutations in DNA help to
uncover the reasons why some people develop certain diseases while others remain
disease free. This discovery and study of DNA helps physicians and researchers
discover how to test for and treat diseases. Understanding DNA also has the potential
to help scientists find a way to produce pharmaceutical drugs that are tailored to an
individual's genetic structure. For further information DNA isolation or DNA estimation
method is used. For that they isolate the DNA. After isolation of DNA, quantification and
analysis of quality, are necessary to ascertain the approximate quantity of DNA
obtained and the suitability of DNA sample for further analysis. This is important for
many applications including digestion of DNA by restriction enzymes or PCR
amplification of target DNA. Forensic science needs to recover DNA for identification of
individuals (for example rapists, petty thieves, accident, or war victims), paternity
determination, and plant or animal identification. One reason DNA extraction is
important is that it makes DNA testing possible in the case of criminal investigations.

Method for DNA Estimation

Material required: 5% Trichloro acetic acid (TCA), 3:1 alcohol ether solution, 0.1 N
KOH solutions, 6N Hydrochloric acid (HCl), 10% Trichloro acetic acid (TCA),
diphenylamine, glass pipette, test tubes etc.

Principle:

This procedure involves chemical hydrolysis of DNA, when heated (e.g. ≥95 °C)
in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA.
Under these conditions, the 2-deoxyribose is converted to hydroxylevulinylaldehyde,
which reacts with the compound, diphenylamine, to produce a blue-colored compound.
DNA concentration can be determined measuring the intensity of absorbance of the
solution at the 620 nm with a spectrophotometer.
Procedure:

DNA ESTIMATION

 For the estimation of DNA, a known amount of tissue i.e 100mg is used.
 For weighing 100mg tissue we require a balance.
 For this a piece of aluminum foil is kept on the pan of the balance.
 By pressing tare the weight of aluminum foil is made zero.
 Now a piece of tissue is cut from the bigger piece in the Petridis and put on the foil in
the balance to weigh 100mg tissue.
 Now take out 100mg tissue carefully along with the foil, pack it and label it as Test 1.
 The same process is repeated.
 Label the 2nd pack as Test 2.
 Now open the packets.
 Put the tissue in already chilled motor.
 For homogenate the tissue by using the pestle by moving pestle in clock wise and
anti clock wise directions.
 Tissue homogenate should always be made carefully.
 Homogenization should be in such a way that tissue particle should not be seen.
 In this grinding process, initially tissue paste is made without any solution.
 Once tissue homogenate is made.
 To this add 5ml of cold 5% (Trichloro Acetic Acid) TCA using a pipette in the motor.
 Mix it with the pestle by rotating the pestle.
 Transfer this homogenate in labeled centrifuge glass tubes as test-1 & test-2.
 Keep these tubes for 30 minutes at 4oC in refrigerator.
 After 30 minutes take out the test tubes from the refrigerator.
 Then transfer the test tubes to a centrifuge.
 Set the time at 5 minute and adjust the rpm at 2000 than close the door and
centrifugation starts.
 After 5 minutes take out the test tubes without disturbing the pellet and place it in the
stand.
 In the tube we can see the supernatant above and pellet is formed at the bottom.
 Gently discard the supernatant in discardant one by one.
 Now take glass pipette and add 5 ml of cold 5% TCA solution in test tube labeled as
test-1.
 Use the same volume of 5% TCA for the test-2.
 Pellet has to be disturbed; using this glass rod and mixes it well.
 After finishing in first test-1 test tube wash the glass rod by D.W. than wipe it with
tissue paper and use for second test-2 test tube to disturbed the pellet.
 Leave the tube for 30 minutes in refrigerator.
 After 30 minutes take test tubes from the refrigerator.
 Now centrifuge both test tubes in centrifugation machine for 5 minutes at 2000 rpm.
 After 5 minutes take out the test tubes without disturbing the pellet and place it in the
stand.
 In the tube we can see the supernatant above and pellet at the bottom.
 Gently discard the supernatant in discarded and pellet remains in the test tube.
 Now 5ml of 3:1 alcohol ether solution is added to the pellet in both the test tubes and
crushed gently with glass rod and mixed properly to get uniform solution.
 After finishing in first test 1 test tube wash the glass rod by distilled water, then wipe
it with tissue paper.
 The test tubes are kept in the BOD incubator for 30 min at 50 0 C temperature.
 Now put the tubes for centrifugation for 5min at 2000rpm.
 Next take out the tubes from centrifuge, observe the pellet formed at the bottom of
the tube.
 Now the supernatant is discarded which contains lipids.
 Same procedure is repeated once more to make the residue completely free of
lipids.
 Now 5ml of 0.1N KOH solution is added to the pellet in the tubes.
 Again disturb the pallet gently with the glass rod and mix it properly and put back the
glass rod after rinsing with distilled water and cleaning with tissue paper.
 Now the tubes are kept in the BOD incubator for 16-18 hrs at 370C.
 With this the first part of the DNA estimation is completed.