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4/6 year 2012/13 NUCLEIC ACIDS
NUCLEIC ACIDS
BEFORE THE LAB YOU HAVE TO READ ABOUT:
1. Structure of the purine and pyrimidine bases in DNA and RNA, nucleosides, and
nucleotides.
2. Oligonucleotides structure (ribo- and 2`-deoxyribo-), full and abbreviated form.
3. Nucleosome structure, histones and their role in nucleosomes.
4. Basic features of RNA and DNA structure.
5. Methods of DNA isolation and analysis.
6. Main differences between pro- and eukaryotic genome organization.
INTRODUCTION
GENERAL CHARACTERISTICS OF EUKARYOTIC
AND PROKARYOTIC GENOMES
In most bacterial cells, genes are encoded on large circular chromosomes. Bacterial chromosome
occurs in the cell as a compact nucleoid structure separated from the cytoplasm. Several mechanisms
operate to compact prokaryotic DNA sufficiently to fit it inside the bacterial cell. For example, the
1 mm-long DNA molecule of the E. coli is packed in the cell, which is about
2 μm long and about 0.5-1 μm wide. The large volume filled by free DNA is due largely to charge
repulsion between the negatively charged phosphate groups. This effect is counteracted by association
of the DNA with positively charged polyamines and with numerous small protein molecules
associated with the bacterial DNA, causing it to fold into a more compact structure. The most
abundant of these proteins, histone-like nucleoid structural protein (H-NS), is a dimer of a 15.6-kDa
that binds DNA tightly. There are about 20 000 H-NS molecules per E. coli cell (one H-NS dimer per
about 400 base pairs of DNA). Bacterial enzyme called DNA gyrase uses energy from ATP hydrolysis
to wind supercoils into DNA. Supercoiling contributes to the compaction necessary to fit
chromosomal DNA into the bacterial cell.
The problem of compacting genomic DNA to fit into the nucleus of eukaryotic cell has been solved
in a different way. When the DNA from eukaryotic nuclei is isolated in isotonic buffers (0.15 M KCl),
it is associated with an equal mass of protein in a highly compacted complex called chromatin. The
most abundant proteins associated with eukaryotic DNA are histones, a family of basic proteins
present in all eukaryotic nuclei. The five major types of histone proteins − termed H1, H2A, H2B, H3,
and H4 − are rich in positively charged basic amino acids: Arg and Lys, which interact with the
negatively charged phosphate groups in DNA. The basic amino acid side chains of histones can be
modified by post-translational addition of acetyl (CH3COO ), phosphate, or methyl groups,
neutralizing the positive charge of the side chain or converting it to a negative charge. The amino acid
sequences of four histones (H2A, H2B, H3, and H4) are remarkably similar among distantly related
species. The amino acid sequence of H1 varies more from organism to organism than do the sequences
of the other major histones. Although histones are the predominant proteins in chromosomes, nonhistone proteins are also involved in organizing the structure of chromosomes.
When chromatin is extracted from nuclei at low salt concentration, it resembles "beads on a string".
In this extended form, the string is a thin filament of "linker" DNA connecting the bead-like structures,
termed nucleosomes. Composed of DNA and histones, nucleosomes are about 10 nm in diameter and
are the primary structural units of chromatin. The DNA component of nucleosomes is much less
susceptible to digestion than is the DNA linker between them. If the nuclease treatment is carefully
controlled, all the DNA linkers can be digested releasing individual nucleosomes. A nucleosome
comprises a protein core with DNA wound around it. The core is an octamer containing two copies of
each of histones H2A, H2B, H3, and H4. Nucleosomes from all eukaryotes contain about 146 base
pairs of DNA wrapped slightly less than two turns around the protein core. The length of the DNA
linker varies among species, ranging from about 15 to 55 base pairs.
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If chromatin is isolated at physiological salt concentration (0.15 M KCl), most chromatin appears as
fiberlike form that is 30 nm in diameter. In these condensed fibers, nucleosomes are thought to be
packed into solenoid arrangement, with six nucleosomes per turn. Chromatin is further organized into
large units, hundreds to thousands of kilobases in length, called chromosomes. The general belief is
that each of the several chromosomes in eukaryotic cells contains a single long DNA molecule. The
longest DNA molecules in human chromosomes are almost 10 cm long (2-3 × 108 base pairs).
The chromatin in chromosomal regions that are not being transcribed exists predominantly in the
condensed, 30-nm fiber form. The regions of chromatin actively being transcribed are thought to
assume the extended beads-on-a-string form. The N-termini of histon proteins contains several
positively charged lysine groups that undergo reversible acetylation and deacetylation by enzymes that
act on specific lysines in the N-termini of the different histones. In the acetylated form, the positive
charge of the lysine amino group is neutralized and the interaction of histones with a DNA phosphate
group is eliminated. Thus, the greater extent of acetylation of histone N-termini, the less likely
chromatin condensation to 30-nm fibers and possibly higher-order folded structures.
PLASMIDS AND RESTRICTION ENZYMES
Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separated from a
chromosomal DNA. These extra-chromosomal DNAs, which occur naturally in bacteria, yeast, and
some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their host cell.
Plasmids range in size from a few thousands base pairs (bp) to more than 100 kilobases (kb). Like the
host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division.
Many naturally occurring plasmids contain genes that provide some benefits to the host cell,
fulfilling the plasmid's portion of the symbiotic relationship. For example, some bacterial plasmids
encode enzymes that inactivate antibiotics. Such drug-resistance plasmids have become a major
problem in the treatment of a number of common bacterial pathogens.
The plasmids most commonly used in recombinant DNA technology replicate in E. coli. Generally,
these plasmids have been engineered to optimize their use as vectors in DNA cloning. For instance, to
simplify working with plasmids, their length is reduced; many plasmid vectors are only around 3 kb in
length (pUC 19 – 2686 bp, pBR-322 – 4362 bp), which is much shorter than in naturally occurring E.
coli plasmids.
Most plasmid vectors contain the essential nucleotide sequences required for their use in DNA
cloning: a replication origin, a drug-resistance gene, and a region in which exogenous DNA fragments
can be inserted. The process of DNA insertion is possible thanks to the restriction enzymes.
Restriction enzymes are bacterial enzymes that recognize specific 4- to 8-bp sequences (restriction
sites), and cleave both DNA strands at this site. Since these enzymes cleave DNA within the molecule,
they are also called restriction endonucleases to distinguish them from exonucleases, which digest
nucleic acids from an end.
Many restriction sites, like the EcoRI site shown in Fig. 1, are short inverted repeated sequences;
that is, the restriction-site sequence is the same on each DNA strand when read in the 5’→3’direction.
Fig. 1. DNA cleavage generated by restriction endonuclease EcoRI.
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Because the DNA isolated from an individual organism has a specific sequence, restriction enzymes
cut the DNA into a reproducible set of fragments called restriction fragments. Depending on the type
of restriction enzyme used, restriction digestion creates DNA fragments with “blunt” or “sticky” ends.
As shown in Fig.1, EcoRI makes staggered cuts in the two DNA strands. Many other restriction
enzymes make similar cuts, generating sticky fragments that have a single-stranded "tail" at both ends.
The tails on the fragments generated at a given restriction site are complementary to those on all other
fragments generated by the same restriction enzyme. At room temperature, these single-stranded
regions can transiently base-pair with those on other DNA fragments generated with the same
restriction enzyme, regardless of the source of the DNA. This base pairing of sticky ends permits DNA
from widely differing species to be ligated, forming chimeric DNA molecules.
EXPERIMENTS
1. DNA extraction from calf thymus
Principle of the method
Methods used for DNA extraction from the biological material aim to obtain chemically pure and
undamaged preparation. During DNA purification, the nuclear proteins and RNA are removed. To avoid
DNA degradation, the endogenous deoxyribonucleases activities should be limited. The high nucleases
activity is characteristic for spleen or pancreas, but it is relatively low in thymus. DNA content in different
tissues varies from 0.2% in rat liver to 2.5% in calf thymus. Moreover, RNA/DNA ratio is about 4 for rat
liver, and only 0.4 for calf thymus. These criteria make the calf thymus appropriate tissue for DNA
extraction.
DNA isolation is a two-steps procedure:
1. Deoxyribonucleoproteins (DNPs) isolation from thymus.
Thymus is homogenized in SSC solution. Homogenization process destroys cells and releases DNP
and RNP from the nucleus. During homogenization RNP is dissolved but DNP remains insoluble.
The sample is free from ribonucleoproteins (RNPs) contamination. Citrate is used to bind (chelate)
divalent ions, i.e. Ca2+ and Mg2+ necessary for deoxyribonucleases action. These ions removal
protects DNA from degradation by nucleases.
2. DNP dissociation.
DNP dissolves in high ionic strength solution (2 mol/l NaCl solution). Dissolved DNA, free from
proteins contamination, is precipitated with ethanol.
Materials and reagents
1. SSC solution (0.15 mol/l NaCl, 0.015 mol/l sodium citrate, pH 7.0)
2. NaCl solution (4 mol/l)
3. Calf thymus
Procedure
1 a) Steps common for all students
1. Homogenize 1g of calf thymus (fresh or frozen) in knife homogenizator for 2 min in 150 ml SSC
solution.
Before centrifugation, buckets with tubes should be balanced in pairs!!!
2. Centrifuge the homogenate (15 min, 2500 rpm).
3. Discard the supernatant containing dissolved RNP.
4. Transfer the DNA sediment to the homogenizator dish.
5. Add 200 ml SSC solution and homogenize for 30 sec to obtain homogenous suspension.
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1 b) Continue the next steps in pairs
6. Pour 10 ml of DNA homogenate to the beaker.
7. Add 10 ml of 4 mol/l NaCl solution (final concentration 2 mol/l).
8. Mix it gently until the solution becomes viscous (sticky) because of DNP dissolving.
Obtained DNP lysate will be used immediately for experiment 2 and 3.
2. Comparison of nucleic acids sensitivity for alkaline hydrolysis
Principle of the method
Alkaline hydrolysis of RNA results in formation of the mixture of 2’- and 3’- ribonucleoside
monophosphates. Cyclic 2’,3- nucleoside monophosphate intermediates are the necessary step to
complete RNA hydrolysis. The released nucleotides dissolve in HClO4 solution and came through the
filter to filtrate. UV absorption confirms the nucleotides presence in the place where RNA hydrolysate
was dropped.
DNA is not hydrolyzed in the alkaline solution. Cyclic intermediates with 2’,3’ - fosfodiester bonds
can not be formed because of the absence of the hydroxyl group at 2’-carbon of deoxyribose. In the
alkaline solution DNA undergoes denaturation, hydrogen bonds between complementary nitrogen
bases are broken and single stranded DNA is formed. In KOH solution, DNA denaturates and stays on
the filter during filtration.
Reagents
1.
2.
3.
4.
5.
6.
0.3 mol/l KOH solution
RNA sample obtained from baker`s yeast
DNP lysate
30% HClO4 solution
pH test papers
96% ethanol
Procedure
1. Pour 10 ml of the DNP lysate (from calf thymus) into the beaker of 50 ml.
2. Add equal amount of cooled 96% ethanol.
3. Mix it gently until clearly visible, flocular sediment of DNA is formed (do not use glass rod).
4. Using a glass rod, transfer precipitated DNA to a dry centrifuge glass tube and add 2 ml of 0.3
mol/l KOH solution.
5. In parallel, add 2 ml of 0.3 mol/l KOH solution to the tube with RNA sample.
6. Incubate both tubes in the water bath at 37C for 1 hour.
7. Using 30% HClO4 adjust pH of cooled DNA and RNA solutions carefully to the value of 3-4.
Verify pH changes with pH test papers. Observe changes in both tubes during acid addition.
8. Filter both solutions separately through filter paper to the new tubes.
9. Put a drop of filtrate on the filter paper, dry it with a hairdryer and repeat 3 times. Visualize in
the UV light and compare the UV absorption intensity of both spots.
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3. Detection of nucleic acids components in acidic hydrolysates of DNA and RNA
Principle of the method
In the presence of strong acids and at elevated temperature, RNA degrades to ribonucleoside
monophosphates and, subsequently, to free nitrogen bases, ribose residues and orthophosphate acid. βN-glycosidic bonds in DNA between sugar and base moieties are hydrolyzed in the acidic solution and
free bases are released. After extended hydrolysis, phosphates and deoxyribose can be formed.
3 a) Preparation of acidic DNA and RNA hydrolysate
Reagents
1.
2.
3.
4.
RNA obtained from baker`s yeast
DNP lysate
0.2 mol/l H2SO4 solution
96% ethanol
Procedure
Turn on the water bath before experiment!
1. Pour 10 ml of the DNP lysate (from calf thymus) into the beaker of 50 ml.
2. Add equal amount of cooled 96% ethanol.
3. Mix it gently until clearly visible, flocular sediment of DNA is formed (do not use a glass rod).
4. Transfer precipitated DNA using a glass rod to a dry centrifuge glass tube and add 2 ml of 0.2
mol/l H2SO4 solution.
5. In parallel, add 2 ml of 0.2 mol/l H2SO4 solution to the tube with RNA sample.
6. Incubate both tubes in the boiling water bath (100C for 30 min).
7. After hydrolysis is completed, make qualitative analysis of nucleic acids components.
3 b) Purines detection
Purines form insoluble salts with silver ions
Reagents
1.
2.
3.
4.
0.5% adenine solution
4 mol/l NaOH solution
25% ammonia solution
5% AgNO3 solution
Procedure
1. Prepare 3 glass tubes.
2. Add 0.5 ml of acidic RNA hydrolysate to the first tube, 0.5 ml of acidic DNA hydrolysate to the
second one, and 0.5 ml of adenine solution to the third one.
3. Add 2-3 drops of 4 mol/l NaOH solution to each tube to make environment alkalic.
4. Add few drops of 25% ammonia solution and few drops of 5% AgNO3 solution to each tube.
5. White, jelly-like sediment of silver salts of purines becomes visible.
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3 c) Phosphate groups detection
Phosphate ions react with ammonium molybdate and form yellow sediment of ammonium
phosphomolybdate – (NH4)3P(Mo3O10)4, which is reduced by metol to “molybdenic blue” (mixture of
molybdenium oxides at lower oxidation state).
Reagents
1.
2.
3.
4.
1% Na2HPO4 solution
Concentrated H2SO4
5% ammonium molybdate solution
2% metol solution (in 5% Na2SO3)
Procedure
1. Prepare 3 glass tubes.
2. Add 0.5 ml of acidic RNA hydrolysate to the first tube, 0.5 ml of acidic DNA hydrolysate to the
second one, and 0.5 ml of 1% Na2HPO4 solution to the third one.
3. Add 1 drop of concentrated H2SO4 and few drops of 5% ammonium molybdate solution to each
tube, mix and add few drops of 2% metol solution. Observe blue or green-blue color.
3 d) Ribose detection
During heating with concentrated HCl in the presence of Fe3+, ribose is dehydrated into furfural, which
forms a colored complex with orcine.
Reagents
1. concentrated HCl (containing Fe3+)
2. Orcine in subst.
Procedure
1. Prepare 2 glass tubes.
2. Add 0.3 ml of acidic RNA hydrolysate to the first tube, and 0.3 ml of acidic DNA hydrolysate to
the second one.
3. Add 1 ml of concentrated HCl (containing Fe3+) and few crystals of orcine to each tube.
4. Boil both tubes. Solution containing ribose changes the color into green.
3 e) Deoxyribose detection
During heating deoxyribose forms a colored complex with diphenylamine.
Reagents
1. Concentrated H2SO4
2. 0.3% diphenylamine solution in CH3COOH
Experimental procedure
1. Prepare 2 glass tubes.
2. Add 0.5 ml of acidic RNA hydrolysate to the first tube, and 0.5 ml of acidic DNA hydrolysate to
the second one.
3. Add 2-3 drops of concentrated H2SO4 and 1 ml of diphenylamine solution to each tube.
4. Heat both tubes. The solution containing deoxyribose changes color into violet-blue. Ribose
reacts with diphenylamine, however the intensity of colour is very low.
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