Hydrolysis of nucleic acids by either chemical or enzymatic methods yield purine or pyrimidine bases, oligonucleotide containing up to 20 residues, nucleosides, ribose or deoxyribose and phosphates.
Acid hydrolysis cleaves susceptible purine N-glycosyl bonds in both DNA and RNA. When RNA is boiled in dilute acid, adenine and guanine are released, leaving an “apurinic acid” which maybe further hydrolyzed to a mixture of pyrimidine nucleotides. The pyrimidines are more resistant to acid hydrolysis. Bond cleavage of the N-glycosyl bonds requires more vigorous conditio0ns like heating with acids in an autoclave or sealed tubes. This would release cytosine and uracil. However, during the process, there is a tendency for cytosine to be deaminated to uracil. Significant hydrolysis of RNA can be obtained by treatment with 1N HCl at 100oC for 1 hour.

            Base hydrolysis of RNA produces a mixture of 2’ and 3’ nucleotides of cyclic 2’, 3’-monophosphate intermediates. These are further hydrolyzed by alkali, which attacks either one of the two P-O-C linkages, to yield a mixture of 2’ and 3’ nucleoside monophosphates. DNA, on the other hand, is not readily hydrolyzed by dilute alkali because it lacks the 2’ hydroxyl group and therefore, cannot form the necessary 2’-3’ cyclic monophosphate intermediates.

            Nucleic acids may be hydrolyzed enzymatically by nucleases, which demonstrate different specificities: sugar specificity, phosphodiester bond specificity, and other specificities.
 

Qualitative Analysis

            Chemical determination of nucleic acids is based on the presence of phosphorous, pentoses (ribose 0r deoxyribose) or purine and pyrimidine base. A brief description f the qualitative tests for these components is given below:

1) Bebedicts’ Test for reducing sugar. (See Experiment 8 of the lab manual: Experiments in
2) Biochemistry, 1998 edition)
3) Orcinol’s Test for pentoses> (See Experiment 2)
4) Test for Purine bases:
5) Free pyrimidines and purine bases are relatively insoluble in water. They are weakly basic compounds that may exist in two or more tautomeric forms depending on the pH. They are precipitated by ammoniacal silver nitrate as their silver salts, which appear as flocculent, white precipitates.
6) Test for Inorganic Phosphates. (See experiment 8 of the lab manual: Experiments in Biochemistry, 1998 ed)
 
 

Top 10 Facts for DNA Electrophoresis

• When preparing agarose for electrophoresis, it is best to sprinkle the agarose into room temperature buffer, swirl, and let sit at least 1 min before microwaving. This allows the agarose to hydrate first, which minimizes foaming during heating.
• Electrophoresis buffer can affect the resolution of DNA. TAE(Tris-Acetate-EDTA) buffer provides better resolution of fragments >4 kb, while TBE(Tris-Borate-EDTA) buffer provides better resolution of 0.1 to 3-kb fragments. In addition, use TBE buffer when electrophoresing >150 V and use TAE buffer with supercoiled DNA for best results.
• Migration of DNA is retarded and band distortion can occur when too many buffers cover the gel. The slower migration results from a reduced voltage gradient across the gel.
• Loading DNA in the smallest volume possible will result in sharper bands
• You can preserve DNA in agarose gels for long term storage using 70% ethanol.
• Electrophoresing a gel too “hot” can cause the DNA to denature in the gel. It can also cause the agarose gel to deform. Cool the gel with a small fan during the electrophoresis.
• For the Supercoiled DNA Ladder electropghoresed on < 1%agarose gels, add 2g/ml ethidium bromide to the gel. Otherwise, smeared bands and extra bands will be seen because of different degrees of supercoiling.
• When glycerol-containing loading buffers are used in DNA samples electrophoresed through acrylamide gels, smiling bands may be accentuated especially in TBE.
• On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than the same size. This anomalous migration is not observed on agarose gels.

The minimum  amount of DNA detectable by ethidium bromide on a 3-mm thick gel and 5-mm-wide lane is 1 ng. Do not exceed 50 ng of DNA per band on a 3-mm-thick gel and 5-mm-wide lane.

Reagents and Supplies

From the lockers and store room :
10% H2SO4
0.3N and 0.05%NaOH
N and 12N HCl
Na2CO3
1N and 12N HCl
 Na2CO3
 Benedict’s Reagent
Orcinol Reagent
6N NH3
Red litmus paper

Things to bring:
4 marbles
glass slides and 6 cover slips

Equipment:
boiling water bath
centrifuge
top loading balance
microscopes

Procedure

Hydrolysis
 
 

                                        1                                 2                             3                             4                           5                      6
                                             1ml DNA +                 1ml DNA +              pinch RNA +            pinch RNA +         1ml DNA            1ml RNA
                                            1mL 1M HCl           1mL 3M NaOH       10ml 10%H2SO4         1ml 1M HCl
 

1. Cover test tubes 1-4 with a marble, and place them in boiling water bath for I hour.

2. Set aside test tubes 5-6 for the qualitative tests (procedure B)

3. Run each of the qualitative tests on the hydrolysates (test tubes 1 to 4), and the unhydrolyzed samples (test tubes 5 to 6).
 
 

Qualitative tests

        For each of the qualitative test, you will need a new set of test tubes, labeled 1 to 6.
 

Test for reducing sugars

•  Neutralize 5 drops of the hydrolyzates with solid Na2CO3. To 2 drops of the neutralized hydrolyzates, add 4 drops of Benedict’s reagent. Likewise, add  4 drops Benedict’s reagent to 2 drops of the unhydrolyzed samples.
•  Heat the mixture in a boiling water bath until brick-red precipitates form or until no further change occur.

Test for pentoses

• To a new set of 6 test tubes, correspondingly put 2 drops of the samples from the test tubes in Procedure A.
• To test tubes 5 and 6, add 1 drop of 12N HCl.
• To all the test tubes, add 4 drops of Orcinol reagent.
• Heat the test tubes in boiling watre bath for 3 minutes until a greenish solution forms or until no further change is observed.

Test for purine bases

•  Treat 5 drops of samples from Procedure A with 6N NH3 until alkaline to litmus
•  Add few drops of 5% AgNO3
•  If no precipitation is obtained, let the solution stand undisturbed for 5 minutes
•  Observe the crystals under a microscope

Test for inorganic phospate

• Add excess NH3 to 5 drops of the samples from procedure A.
• Acidify with 6N HNO3
• Add 5 drops ammonium molybdate raegent
• Warm in a water bath for 10 minutes and observe for precipitate formation. If no visible result is observed, add more of the samples

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Chemistry 145.1
Human Genome Project and Bioinformatics
Experiment No. 1
Experiment No. 2
Experiment No. 3
Experiment No. 4
Experiment No. 5
Experiment No. 6
Experiment No. 7
Experiment No. 8
Experiment No. 9
Experiment No. 10
Experiment No. 11
Experiment No. 12
References