Nucleic Acid Analysis Using Bials and Dische Reaction and Gel Electrophoresis'

1
Nucleic Acid Analysis Using Bials and Dische
Reaction and Gel Electrophoresis.
Dan Eltringham, Suzanne Khalafalla, Muriel
Langley, Adam Satteson, Courtney Veeck

• Bials Test
• ?Combine 5µg of nucleic acid, 60µL orcinol acid
  reagent, and 40µL of 6 alcoholic orcinol in a
  0.2mL microcentrifuge tube. Repeat twice.
• Combine 60µL orcinol acid reagent, 40µL of 6
  alcoholic orcinol, and 10µL sterile deionized
  water in a 0.2mL microcentrifuge tube. Repeat
  twice.
• Combine 5µg nucleic acid and 100µL sterile
  deionized water in a 0.2mL microcentrifuge tube.
  Repeat twice.
• ?Mix each tube and place in a thermal cycler at
  94?C for 20 minutes and observe tubes.
• Dische Test
• Combine 5µg nucleic acid, 20µL sterile deionized
  water, and 60µL diphenylamine reagent in a 0.2mL
  microcentrifuge tube. Repeat twice.
• Combine 30µL sterile deionized water and 60µL
  diphenylamine reagent in a microcentrifuge tube.
  Repeat twice.
• Combine 5µg nucleic acid and 80µL sterile
  deionized water in a microcentrifuge tube. Repeat
  twice.
• Mix each tube and place in a thermal cycler at
  94?C for 10 minutes and observe tubes.
• S1 Nuclease
• In a 125mL Erlenmeyer flask add 0.5g agarose and
  50mL 1XTAE.
• ?Microwave for 1.5 minutes or until agarose is in
  solution. Swirl the flask gently every 15 seconds
  to prevent boiling over in the microwave.
• Add 5µL EtBr solution to the solution.
• Pour the gel into the electrophoresis chamber
  containing the eight -welled comb and allow to
  mold for 30 minutes.
• Add 1µg nucleic acid, 1 unit enzyme, 2µL enzyme
  buffer, and sterile deionized water to a total of
  20µL in a 0.2mL microcentrifuge tube. Repeat
  twice.
• ?Add 1µg definite double stranded DNA, 1 unit
  enzyme, 2µL enzyme buffer, and sterile deionized
  water to a total of 20µL in a 0.2mL
  microcentrifuge tube. Repeat once.
• ?Add 1µg definite single stranded DNA, 1 unit
  enzyme, 2µL enzyme buffer, and sterile deionized
  water to a total of 20µL in a 0.2mL
  microcentrifuge tube. Repeat once.
• Add 1µg nucleic acid, 2µL enzyme buffer, and
  sterile deionized water to a total of 20µL in a
  0.2mL microcentrifuge tube.

Abstract In Virulent, Pa thirty students were
infected with a mysterious Illness with symptoms
including nausea, vomiting, fever and muscle
aches. There were four viruses the local
physicians had narrowed it down to the
Parvovirus, the Reovirus, the Orthomyxovirus, and
the Papovaviridae virus. Nucleic acid was
extracted from the children to be tested to
analyze the structural properties in order to
determine the correct virus. Performing the
Bials and Dische tests on the nucleic acid would
determine if it was DNA or RNA. The endonucleic
test performed on the nucleic acid then
determined if it was double stranded or single
stranded. The results showed that the sample was
double stranded DNA. With this data the virus was
best identified as Papovaviridae.

• Discussion Conclusion
• The results of Table 1 show that there was no
  reaction in the Bials test resulting in color
  change. This indicated that the sample was not
  RNA.
• ?In addition, Table 1 shows that there was a
  reaction in the Dische test resulting in a blue
  color change. This indicated that the sample was
  DNA.
• The DNA was broken down into the component
  pieces, including hydroxylevulinyl aldehydes,
  which reacted with the diphenylamine and formed
  the blue color.
• Both the Bials and Dische tests had controls.
• ?The orcinol acid reagent, 6 alcoholic orcinol,
  and water was a control to show that these
  compounds would not react without the nucleic
  acid.
• Likewise, the control with nucleic acid and
  water was present to show that the nucleic acid
  would not react with the orcinol acid and 6
  alcoholic orcinol.
• In the Dische test the controls were present to
  show that the diphenylamine reagent and water
  would not cause a color change without nucleic
  acid.
• Likewise, the control with nucleic acid and
  water was present to show that a reaction would
  not occur without diphenylamine reagent.
• In each microcentrifuge tube, water was added to
  keep the volume constant. This was another
  important control.
• The S1 nuclease was used to break down the
  phosphodiester bonds and determine that our
  nucleic acid was double stranded DNA.
• In Table 2, the unknown sample was not cleaved
  by the S1 nuclease and was therefore concluded to
  be double stranded.
• The single stranded control was used to compare
  our sample. The extreme digestion caused the very
  small fragments of single stranded DNA to travel
  through the gel matrix and into the buffer
  solution.
• ?The control in well 8 was performed to provide
  us with information about how well the enzyme was
  cleaving, verifying that the nucleic acid wasnt
  cleaving itself.
• ?The only virus in the list of possibilities that
  was double stranded DNA was Papovaviridae.
• ?A curious result was that the presence of warts,
  a key symptom, was not included in the
  descriptions of the childrens illness. This is
  possibly because they have not yet formed.
• No significant error was detected in this
  experiment.
• Possible future experiments include taking a
  second sample from the infected children and
  examining it under a microscope to compare the
  structure of the virus for confirmation of the
  viral identification.

Table 2. The results from the S1 nuclease test.
The results of the gel electrophoresis with the
unknown nucleic acid, double and single stranded
controls, and a control with no enzyme. The bands
of the unknown matched the bands on the definite
double stranded DNA control. There were no bands
present at the single stranded DNA wells. This
led to the conclusion that the unknown was double
stranded.
Introduction The Bials Reaction uses alcoholic
orcinol to break down RNA into purines,
pyrimidines and ribose sugars. Those ribose
sugars then dehydrate and react with orcinol and
produce a blue-green color. The acid is not
capable of breaking down the DNA, which allows
the color to stay yellow. The Dische Reaction
uses diphenylamine to break down DNA into
purines, pyrimidines, and deoxyribose sugars.
The deoxyribose sugars dehydrate and form
hydroxylevulinyl aldehydes. These aldehydes will
react with the diphenylamine and form a blue
color (Tobin-Janzen, 2005). This acid is not
capable of breaking down the RNA. Therefore if
both tests are run on the same sample only one
positive should occur. Another important
factor in determining the structure of the
nucleic acid is to test whether it is double or
single stranded. Cleaving with a restriction
enzyme known as S1 Nuclease separates single
stranded DNA and RNA into smaller subunits, while
not affecting the double stranded DNA. This
occurs because the S1 Nuclease endonucleolytically
disrupts the phosphodiester bonds that hold
adjacent nucleotides together. Double stranded
DNA and RNA are not affected by the breakdown
unless there is an extremely high concentration
of enzyme present or if it is super coiled
(Tobin-Janzen, 2005). Gel electrophoresis uses
an electric field to separate nucleic acids
according to their length. Nucleic acids are
negatively charged due to their phosphate groups.
When the nucleic acids are placed in the
electric field they migrate through an agarose
gel matrix towards the positive end of the field.
The matrix limits mobility and therefore smaller
nucleotides are able to weave through it more
easily and travel further. Agarose Gels come in
different concentrations depending on the
estimated size of the nucleic acids. The larger
the nucleic acids the smaller the concentration
for agarose is preferred. The four possible
viruses the children could have are the
Parvovirus, the Reovirus, the Orthomyxovirus, and
the Papovaviridae virus. The Orthomyxovirus is
single stranded RNA and Papovaviridae contains
double stranded DNA (Buchen-Osmond, 2004).
General symptoms for Papovaviridae include a wide
variety of warts (Shanley, 2005). The Reovirus is
double stranded RNA and the Parvovirus is single
stranded DNA (Rasouli, 2005, Cunningham, 2005).
Hypothesis Bials and Dische If the nucleic
acid is RNA, then the Bials test will show a
color change from light yellow/colorless to a
green/blue. If the nucleic acid is DNA, then the
Dische test will show a color change from
yellow/colorless to green/blue. S1 Nucleus If
the nucleic acid is double stranded then it will
not be digested and will only migrate as far as
the double stranded DNA control in the gel
electrophoresis. If the nucleic acid is single
stranded then it will be digested and travel as
far as the single stranded DNA control in the gel
electrophoresis.
Figure 1. The photograph of the gel
electrophoresis under ultraviolet light. The
well numbers proceed from left to right 1-8. The
major components in each well follow wells 1-3
had the unknown nucleic acid sample wells 4-5
had the definite double stranded DNA wells 6-7
had the definite single stranded DNA well 8 had
nucleic acid without enzyme. The bands of the
unknown sample match the definite double stranded
DNA. No bands appeared from the definite single
stranded wells. Well 8 showed an unevenly formed
thick band.
Figure 2. Two photographs of Papovaviridae under
microscope. The yellow picture shows multiple
Papovaviridae and the black and white shows a
closer image.
Results Table 1. The results of the Bials and
Dische tests. The Bials test resulted in no color
change for any of the solutions. However, the
Dische test with the unknown nucleic acid 3,
water, and diphenylamine reagent resulted in a
blue color change. The other two had no color
change. The color change in the Dische test
indicated DNA. Each test was completed three
times for a total of 18 samples, 9 for Bials and
9 for Dische.
References Buchen-Osmond, Dr. Cornelia. The
Universal Virus Database of the International
Committee on Taxonomy of Viruses. 15 June 2004.
Available http//www.ncbi.nlm.nih.gov/ICTVdb/ICTVd
B/index.htm Cunningham, Dr. Dennis and Dr,
Margaret Rennels. Parvovirus B19 Infection.
Last Updated 29 June 2005. Available
http//www.emedicine.com/ped/topic192.htm Rasouli
, Dr. Gholamreza Dr. John King. Reoviruses.
Last updated 6 July 2005. Available
http//www.emedicine.com/med/topic2007.htm Shanle
y, Dr. John D. Papillomavirus. Last Updated 13
July 2005. Available http//www.emedicine.com/MED/
topic1729.htm Tobin-Janzen, Dr. Tammy C. Module
Mystery One The Case of the Virulent Virus.
Susquehanna University Biology Department. Fall
2005.