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					Life science Study materials                                              Chemistry of Nucleic Acids
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                                  (Basics of Nucleic Acids)




                                                             I.Satish Kumar
                                                              Lecturer in Biochemistry
                                                     Email: Satishkumar7777@gmail.com
                                                     Blog: http://biochemistryden.blogspot.com
                                                           http://Biohunting.blogspot.com




    My Sweet Name:_____________________________________________________________

    Class:_________________ Roll/Admission number: ___________ Year :_______________

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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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                                     Nucleic Acids
Introduction:
          The nucleic acid is the molecular repositories for genetic information. The storage of biological information
is the only known function of DNA and is jointly referred to as molecules of heredity.
          The structure of every protein and every cell constituent is the product of information programmed into the
nucleotide sequence of a cells nucleic acid (DNA).
          The amino acid sequence of every protein and the nucleotide sequence of every RNA molecule in a cell are
separated by that of cells DNA. The necessary protein (or) RNA sequence information is found in corresponding
nucleotide sequence in the DNA.

Historical Aspects of nucleic acids
      •     Frederick meischer (1868) first identified phosphate rich substances in hospital bandages puss cells. He had
            considered phosphate rich substances are called “Nuclein”.
      •     Alberecht Kossel (Nobel prize, 1910) differentiated RNA and DNA in 1882.
      •     In 1906, Kossel described the 4 bases in nucleic acids.
      •     The term “Nucleic acid” was first coined by the scientist “Altmann”. The name given based on two
            characters one is based on the location (Nucleous-“Nucleic”) and nature (Acidic nature-“Acid”). The literal
            meaning is “the acidic phosphate rich substances present in Nucleous”, so the name was given as
            NUCLEIC ACID.
      •     In 1931, Barbara McClintock showed the rearrangement of genes or mobile genes in chromosomes in corn
            (Nobel prize, 1983).
      •     X-ray crystallographic studies on DNA by Maurice wilkins (Nobel prize, 1962) showed he details of
            structure of DNA.
      •     Rosalind Franklin worked out the helical structure of DNA.
      •     James Watson and Francis Crick in 1953 deduced the double helical structure of DNA (Nobel prize 1962).

Nature of Nucleic acids:
            DNA (Deoxyribonucleic acid): Deoxyribonucleic acid, which is the major constituent of the
            chromosome located in the nucleus of the cell, but small amounts are associated with cell organelles such
            as chloroplast and mitochondria. The DNA constituents the genetic material.
            RNA (Ribonucleic acid): Ribonucleic acid functions as working copies of DNA & participate in protein
            biosynthesis in some viruses RNA functions as genetic material.
                                       Eg: TMV

                                      Components of Nucleic Acids
   Nucleotides have three characteristic components. They are:
                          a) Nitrogenous Bases
                                                                        Solubility of Bases:
                          b) Sugar Moiety                                   •     At neutral pH, Guanine is the least
                          c) Phosphorus acid                                      soluble of the bases, followed in this
                                                                                  respect by xanthine.
 a)       Nitrogenous Bases: The nitrogenous bases are the                  •     Although uric acid, as urate is
          derivatives of two parent compounds. They are PURINES                   relatively soluble at a neutral pH, it is
          & PYRIDINES                                                             highly insoluble in solutions with a
                                                                                  lower pH, such as urine.
          Purines: Purine bases are heterocyclic compounds                  •     Guanine is not a normal constituent
          consisting of a pyrimidines ring and an imidazole ring                  of human urine, but Xanthine and
          fused together. The two purine bases are-                               Uric acid occur in human urine.
                                                                                  These two purines frequently occur as
                  • Adenine (6-Amino Purine): (C5H5N5),                           constituents of urinary tract stones.
                      found in both RNA and DNA, is a white                       E.g.: Xanthine stones/ and Urate
                      crystalline purine base, with Molecular weight              Crystals.
                      135.15 daltons and melting point 360 to
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Life science Study materials                                              Chemistry of Nucleic Acids
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                   365oC.
               •   Guanine (2-Amino-6-oxyPurine): (C5H5ON5), also found in both DNA and RNA, is a
                   colorless, insoluble crystalline substance, with MW=151.15 dlatons. It was first isolated from
                   guano (bird manure), hence so named.




          i)        Pyrimidines: Pyrimidine bases consist of six membered ring with two nitrogen atoms. The
                    pyrimidine bases are –
                      •     Cytosine (2-Oxy-4-amino pyrimidine): (C5H6O2N5), found in both RNA and
                            DNA, is a white crystalline substance, with MW=111.12 daltons and a melting point 320
                            to 325OC.
                      •     Thymine (2, 4-dioxy-5-methyl pyrimidine): (C5H6O2N2), found in DNA
                            molecules only, has MW=126.13 Daltons. It was first isolated from thymus, hence so
                            named. Only rarely does thymine occur in RNA.
                      •     Uracil (2, 4-dioxy pyrimidine) (C4H4O2N2), found in RNA molecules only, is a
                            white, crystalline pyrimidine base with MW=112.10daltons and a melting point 338OC.
                            only rarely does uracil occur in DNA.




         2. Sugar moiety:
                 Pentose sugar is present in DNA & RNA. It is present in their “β-furanose” from (close five
         number ring) and of β-configuration. Two types of pentose sugars present in the nucleic acid. I) Ribose
         (RNA) and ii) Deoxyribose (DNA).




         3. Phosphate:
                   It contains the monovalent hydroxyl groups and one divalent
         oxygen atom all are linked to pentavalent phosphorous atom.
                  The base is joined covalently (at N1 of pyrimidines and N9 of
         purines) and the phosphate is esterified to the 5’-carbon. The N-glycosyl
         bond is formed by removal of the elements of water (a Hydroxyl groups
         from pentose and Hydrogen atom from the base).


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Life science Study materials                                              Chemistry of Nucleic Acids
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                                              Nucleosides
                 The nucleosides are compounds in which the nitrogen bases (purines & pyrimidines) are
         conjugated to pentose sugar (2-deoxy ribofuranose) by β-glycosyl linkage.

                               Nitrogenous base + Sugar = Nucleoside
    •    This linkage in purine nucleosides is at position -9 of the purine base and carbon 1’ of sugar or deoxy
         sugar.
    •    In pyrimidine nucleosides, β-glycosidic linkage is formed at position -1 of the pyrimidine base linked to
         carbon -1’ of ribose or deoxyribose sugar.
    •    In cytidine and uridine, ribose is attached to N1-position of cytidine and uracil respectively.

        The nucleosides are generally named for the particular purine or pyrimidine present. Nucleosides
    containing Ribose are called “Ribonucleosides”, while those possessing deoxyribose as
    deoxyribonucleosidases.


                                              Nucleotides
          A nucleotide is a nucleoside to which a phosphoric acid group has been attached to the sugar molecule by
‘esterification’ at a definite –OH group and thus has the general composition “base-sugar-PO4” simply, nucleotides
are the phosphoric acid esters of nucleosides. These occur either in the free form or as subunits in nucleic acids.

                                    Sugar+                   Nucleot
                 Nitrogenous base + Sugar+ Phosphoric Acid = Nucleotide




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   Life science Study materials                                              Chemistry of Nucleic Acids
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Nitrogen    Sugar         Nucleoside          Trival Name         Phosphate       Nucleotid        Trival         Abbrevatio
ous base                                                            group            e             name              ns

Ribonucleosides
Adenine      Ribose          Adenine             Adenosine          +H3PO4        Adenosine-      Adenylic          AMP
                          ribonucleoside                                              5’-           acid
                                                                                  monophosp
                                                                                     hate
Guanine      Ribose          Guanine             Guanosine          +H3PO4        Guanosine-      Guanylic          GMP
                          ribonucleoside                                              5’-           acid
                                                                                  monophosp
                                                                                     hate
Cytosine     Ribose          Cytosine             Cytidine          +H3PO4         Cytidine -     Cytidylic         CMP
                          ribonucleoside                                              5’-           acid
                                                                                  monophosp
                                                                                     hate
Thymine      Ribose          Thymine            Thymidine           +H3PO4        Thymidine-     Thymidilic          TMP
                          ribonucleoside                                              5’-           acid
                                                                                  monophosp
                                                                                     hate
 Uracil      Ribose           Uracil              Uridine           +H3PO4        Uridine-5’-   Uridylic acid       UMP
                          ribonucleoside                                          monophosp
                                                                                     hate

Deoxyribonucleosides
Adenine     deoxyri         Adenine            Deoxyadenosine        +H3PO4      Deoxyaden      Deoxyadenyl         dAMP
             bose      deoxyribonucleoside                                        osine-5’-       ic acid
                                                                                 monophosp
                                                                                     hate
Guanine     deoxyri         Guanine            DeoxyGuanosine        +H3PO4      DeoxyGuan       Deoxyguany         dGMP
             bose      deoxyribonucleoside                                        osine-5’-        lic acid
                                                                                 monophosp
                                                                                     hate
Cytosine    deoxyri         Cytosine            Deoxycytosine        +H3PO4      Deoxycytos      Deoxycytidy        dCMP
             bose      deoxyribonucleoside                                         ine-5’-         lic acid
                                                                                 monophosp
                                                                                     hate
Thymine     deoxyri         Thymine            Deoxythymidine        +H3PO4      Deoxythym      deoxythymid         dTMP
             bose      deoxyribonucleoside                                        idine-5’-       ilic acid
                                                                                 monophosp
                                                                                     hate
 Uracil     deoxyri           Uracil            Deoxyuridine         +H3PO4      DeoxyUridi     deoxyuridilic       dUMP
             bose      deoxyribonucleoside                                         ne-5’-           acid
                                                                                 monophosp
                                                                                     hate




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Life science Study materials                                              Chemistry of Nucleic Acids
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Stability and formation of phospho-diester linkage:
         The         successive
nucleotides of DNA are
covalently linked through
phosphate      group    bridges
specifically the 5’-OH group of
one     nucleotide     by     a
phosphodiester linkage.

          Thus the covalent
backbone of nucleic acid
consist       of     alternating
phosphate and pentose residues
of bases as side group joined to
the back bone of regular
intervals.

          The backbone of
DNA is hydrophilic & the
hydroxyl group of the sugar
residue from hydrogen bonds
with water. The phosphate
groups are covalently ionized
and negatively charged. Thus DNA is an acid. Their negative charges are neutralized generally by ionic interactions
with positively charges particles, metal ions and polyamines. The linear nucleic acid stand has specific polarity and
distinct end 5’ & 3’.



Properties of Nucleic Acids:
    a)   Optical Properties:
              The purine and pyrimidine bases found in nucleic acids strongly absorb UV radiation at 260nm. Which
         is generally 35 to 45% less then the optical density is called “Hyperchromic effect”. This effect is due to
         the presence of closely stacked bases one on top of the other giving rise to a helical structure. This optical
         property is useful for the calculation of degree of helicity of DNA.
    b) Optical Rotation:
            The pyrimidine source of dissymmetry in DNA (a) The sugar component of the nucleotide, (b) The
       (right handed) helical structure. DNA has a large, positive rotation, lying between +100 and +1500. Thus
       the helical structure of DNA must be an important denaturation should cause a drop in optical rotation.
            Upon cooling thermally denatured DNA some restoration of optical rotation takes place, indicating
       “renaturation” of the double helical structure. The effect of heat is to “melt out” tracks of hydrogen bonded
       base pairs, causing a loss of helical character and rotation.

    c)   Viscosity: The viscosity of neutral solutions of DNA is very high, but upon adding salts, the viscosity
         decreases. This may be due to the effect of the negative charge of the phosphodiester grouping which
         undergoes a primary dissociation at low pH. At low ionic strength, repulsion is occurring between negative
         charges and finally the double helix is fully extended. The charge of the phosphate group may be partially
         screened and the helix permitted to take a tighter configuration.
             At higher salt concentration double helix DNA will hydrate. This is due to melting out of regions of
         hydrogen bonded base pairs from the ordered double helical structure.
    d) Optical Density:
            The density of DNA from different sources can be determined by “Density Gradient Centrifugation”.
       In this technique, concentrated “Cesium Chloride” solution is mix with the DNA solution and continues the

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Life science Study materials                                              Chemistry of Nucleic Acids
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         centrifugation up to equilibrium reaches. The G≡C pairs are more compact and of higher density at the
         bottom of the tube and A=T pairs are lightest density at the top of the tube.
    e)   Hyperchromic Effect:
              DNA molecule absorbs light energy. This is a property of individual bases. The intact DNA absorbs
         less energy as its bases are packed into a double helix. A denatured DNA molecule absorbs more light as its
         bases in single strands are exposed.
              Increase in temperature leads to melting of double helix and conversion of it to randomly coiled form.
         Then the hidden bases will be exposed and the optical density will also be increased. This phenomenon is
         called “Hyperchromic Effect”.
              When the native DNA is denatured, there occurs a marked increase in optical absorbancy of UV light
         by pyrimidine and purine bases, an effect called “Hyperchromicity”, which is due to un stacking of the base
         pairs. This change reflects a decrease in hydrogen-bonding. This phenomenon can be used to distinguished
         single (or) double stranded DNA in an unknown sample. The effect is due to the disruption of the electronic
         interaction among the bases.
    f)   Melting Temperature:
               The melting temperature is defined as the temperature at which half the helical structure is lost. The
         two strands of a DNA helix readily separated. When the hydrogen bond between its paired bases are
         disrupted. This can be accomplished by heating a solution of DNA are by adding acid (or) alkali to ionize
         its bases.
              This melting temperature where helix enters into coil form. In this transition as temperature increases
         amide point will each where the DNA remains half in the helix form and half in the coil form. In this
         temperature, the absorbancy will increase.
              The DNA molecules containing less G≡C bond denature first, because these are paired by three
         hydrogen bonds, rising the Tm from 77 to 1000C than A=T bond denature later because it have two
         hydrogen bonds only. As the fraction of G≡C pairs increase from 20% to 70%.

    g) Effect of pH:
           Denaturation of DNA helix also occurs at acidic and alkaline pH values at which ionic charges of the
       substituents on the purine and pyridine bases can occur. In acid solutions near pH 2 to 3, at which amino
       groups bind proteins, the DNA helix is disrupted. Similarly, in alkaline solutions near pH 12, the enolic
       hydroxyl groups ionize, thus preventing the keto-amino group hydrogen bonding.
    h) Absorption Spectra:
             Measurement of light absorption is important for the analysis of nucleotide and the nucleic acids. The
       fraction of incident light absorbed by a solution at a given wave length is related to the “thickness of the
       absorbing layer” and the concentration of the absorbing species. These two relations are combined into the
       “Beer-Lambert law”. That is
                  Log Io/I=€Cl                                       Io = Intensity of the incident light
         Io/I are called “Absorbency is distinguished as “A”.        I= Intensity of the transmitted light
         The molar absorption coefficient varies nature of the       € = Molar absorption coefficient
                                                                     C=Concentration of the absorbing species (Moles/litre)
         absorbing compound, the solvent, the wavelength and
                                                                     l= Thickness of the light absorbing sample
         with pH.




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Life science Study materials                                              Chemistry of Nucleic Acids
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                                    Structure of Nucleic Acids
                       Deoxyribonucleic acid (DNA)
Watson & Crick DNA double helix structure:
       On the basis of X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins and the analytical data of
Chragaff. James Dewey Watson and Francis Harry Compton Crick in 1953 proposed three dimensional double
helical DNA.

Salient features:
      1. It consists of two polyribonucleotide chains trusted
           around one another to form a right handed double
           helix.
      2. The hydrophilic backbones of alternating deoxyribose
           and negatively charged phosphate groups are on the
           outside of the double helix, facing the surrounding
           water.
      3. The purines and pyrimidine bases of both strands are
           stacked inside the double helix.
      4. Each chain is made up of 3’           5’ phosphodiester
           internucleotide linkage.
      5. The two chains are held together in helical shape by
           interchain binding forces such as hydrogen bonds and
           hydrophobic bonds.
      6. The two strands are complimentary & run in opposite direction i.e., if one chain runs from 3’ 5’. Other
           will runs in the direction 5’ 3’ establishing a polarity. Thus the two strands of helix are antiparallel.
      7. In base pairing Adenine pairs with Thymine by two hydrogen bonds and Guanine pairs with Cytosine by
           three hydrogen bonds.
      8. Each turn of helix consists of 10.5 pair of nucleotide residues with an internucleotide space of 3.4A0
           making each helix 34A0 long.
      9. The width of the double helix is 20A0.
      10. The special relationship between the two strands creates a major groove & minor groove.

Different Structural forms of DNA:
                  DNA      is    remarkably     flexible  molecule
         considerable reaction is possible around a number of bonds
         in the sugar-phosphate backbone & thermal fluctuations
         can produce bending, stretching & unpairing in the
         structure.
                  Many deviations from Watson & Crick DNA
         structure are found in cellular DNA & all of these may
         play an important role in DNA metabolism. These
         structural variations generally do not affect the key
         properties of DNA defined by Watson & crick.
    The Watson and crick structure is also defined to as + B-form
    of DNA
    1. B-DNA: this is a normal right handed double helix and
        therefore the standard point of reference in any study of the
        properties of DNA. The two other structural variants of
        DNA are A & Z form.
    2. A-DNA: It is favored in many solutions that are relatively
        devoid of water. It is a right handed double helix. The pitch is reduced from 3.4 to 2.8A0 with 11 base pairs
        / turn.

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    3.   Z-DNA: It is a left handed double helix; these are 12 base pairs per helical turn. The DNA backbone
         appears in a zig-zag model.




Watson & Crick Model of DNA molecule:
The salient features of Watson & crick model of DNA are given below:
    1.   Right handed double helix: DNA consists of two polydeoxy ribonucletide chains twisted around one
         another in aright handed double helix similar to a spiral staircase. The sugar and phosphate groups
         comprise the handrail and the bases jutting inside represent the steps of the staircase. The bases are located
         perpendicular to the helix axis, whereas the sugars nearly at right angles to the axis.
    2.   The base pairing rule: Always the two strands are complementary to each other. So, the adenine of one
         strand will pair with thymine of the opposite strand, while guanine will pair with cytosine. The base pairing
         (A with T; G with C) is called Chargaff’s rule, which states that the number of purines is equal to the
         number of pyrimidines.
    3.   Hydrogen bonding: The DNA strands are held together mainly by hydrogen bonds between the purine and
         pyrimidine bases. There are two hydrogen bonds between A and T while there are hydrogen bonds
         between C and G. the GC bond is therefore stronger than the AT bond.
    4.   Antiparallel: The two strands in a DNA molecule run antiparallel, which means that one strand runs in the
         5’ to 3’ direction, while the other is in the 3’ to 5’ direction. This is similar to a road divided into two, each
         half carrying traffic in the opposite direction.
    5.   Other features: In the DNA, each strand acts as a template for the synthesis of the opposite strand during
         replication process.

        The spiral has a pitch of 3.4 nanometers per turn. Within a single turn, 10 base pairs are seen. Thus, adjust
    bases are separated by 0.34nm. The diameter or width of the helix is 1.9 to 2.9 nanometers. A major groove (1.2
    nm) and a minor groove (0.6nm) wind along the molecule, parallel to the phosphordiester backbone. In these
    grooves, proteins interact with the exposed bases.

    Denaturation of DNA strands:
                 The double stranded DNA may be denatured and separated by heat. This is called melting of
         DNA. Tm or melting temperature is the temperature when half of the helical structure is denatures. At
         lower temperature, the melted strands are reassociated; this is called annealing.




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Life science Study materials                                              Chemistry of Nucleic Acids
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Base composition of DNA:
          A most important clue to the structure
of DNA came from the work of Erwin
Chargaff & his colleagues in 1940’s. They
found that the fair nucleotide bases in DNA
occur in different ratios in the DNA’s of
different organisms.
         There are certain generalizations
regarding the pattern of base composition in
DNA. There are as follows:
a. The base composition of DNA generally
varies from one species to another.
b. DNA isolated from different tissues of the
same species has the same base composition.
c. The base composition of DNA in a given
species does not change with the age,
nutritional status or changing environment.

   In all DNA’s regardless of the species the
number of Adenine residues is equal to the
number of Thymine residues (A=T) and the
number of Guanine residues is equal to the
number of thymine residues (A=T) and the
number of Guanine residues is equal to the
number of Cytosine residues (G=C).

    1.   Adenine always pars with Thymine (in DNA) or Uracil (in RNA) with two hydrogen bonds.
    2.   Guanine always pairs with Cytosine (both in DNA and RNA) with three hydrogen bonds.
    3.   The sum of purine residues equal to sum of pyrimidine residues.
    4.   The ratio of Adenine of Thymine =1 (i.e., A/T=1)
    5.   The ration of Guanine of Cytosine =1 (i.e., G/C=1)
    6.   The A+T/G/C are known as dissymmetry ratio and vary greatly from one species to other. When
         dissymmetry ratio exceeds one such DNA is called AT type & less one such DNA GC type.

  Chargaff’s Rule:
           A most important clue to the structure of DNA came from the work of Erwin Chargaff and his
  colleagues in the late 1940s. They found that the four nucleotide bases of DNA occur in different ratios in the
  DNAs of different organisms and that the amounts of certain bases are closely related. These data, collected from
  DNAs of a great many different species, led Chargaff to the following conclusions:
  1. The base composition of DNA generally varies from one species to another.
  2. DNA specimens isolated from different tissues of the same species have the same base composition.
  3. The base composition of DNA in a given species does not change with an organism’s age, Nutritional state or
  changing environment.
  4. In all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of
  thymidine residues (that is, A=T), and the number of guanosine residues is equal to the number of cytidine
  residues (G≡C).
             From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine
  residues; that is, A + G = T + C.
           These quantitative relationships, sometimes called “Chargaff’s rules,” were confirmed by many
  subsequent researchers. They were a key to establishing the threedimensional structure of DNA and yielded clues
  to how genetic information is encoded in DNA and passed from one generation to the next.




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                                                            Units of length:
                                                            For the measurement of lengths, DNA double
                                                            stranded structure is considered, and expressed in the
                                                            form of base pairs (bp).

                                                                       1 kilobase pairs = 1000bp
                                                                1Mega base pairs = 1000kb =1,000,000bp
                                                              1 Gigo base pairs = 1000 mb = 1,000,000,000




                                       Supercoiling of DNA
         The double helix structure of DNA represents a linear DNA molecule, but DNA in vivo often has a closed
structure without any free ends. In bacteria and viruses, DNA is often circular. In eukaryotes also, large loops of
DNA are found in such a way that each loop represents part of a circle. This organization puts an additional
constraint on double helical structure, and the DNA becomes supercoiled.

         One supercoil is introduced every time that the duplex thread is twisted about its axis. This supercoiling
place a DNA molecule under torsion, which can be released by a break in one of the two strands. A DNA molecule
under torsion, which can be release by a break in one of the two strands. A DNA molecule without supercoiling is
said to be relaxed.

         The supercoils may be negative (as found in vio), when they are in a direction opposite to the clockwise
turns of right handed DNA. This will become under wound or even single stranded in a region. This helps in
unwinding of DNA for replication, etc. the positive supercoils lead to over winding (super helical), which can be
created in vitro, but does not occur in nature.

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         Supercoiling is often controlled by some enzymes and is described by parameters like linking number,
twisting number and writhing number. Supercoiling enzymes are Topoisomerases.

Topoisomerases can be of type I (making transient break in one strand) or type II (making transient double strand
breaks).

    • Type I Topoisomerases (e.g.: product of top A gene of E.coli) relax negatively supercoiled DNA, while
    • Type II (e.g: gyrase) relax both negative and positive supercoils,.
    The removal of supercoils from the crossing segments is required during the recombination process.

Supercoiling are two types:
        a)          Positive super coiling: It involves the coiling of double helix. In some direction of the turns of
                    the two strands of double helix.
        b)          Negative super coiling: It involves the coiling of above double helic\x in the direction opposite
                    to the turns of the strands.




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                     Ribonucleic Acids (RNA)
        Ribonucleic acid (RNA), like DNA, is a long, unbranched macromolecule consisting of nucleotide
joined by 3’ 5’ phosphodiester bonds. The number of ribonucleotides in RNA ranges from as few as 75
to may thousands.

         The second major form of nucleic acid in cells is RNA. It plays the role of intermediary in converting this
information into a functional protein. RNA is found both in nucleus & cytoplasm. The RNA carries the genetic
information from DNA to the protein biosynthetic machinery of the ribosome.
         The RNA is single stranded but at certain regions it may be folded upon itself to form double stranded
regions. The repeating unit of RNA is ribonucleotide which is composed of phaosphate, ribose sugar & nitrogenous
base. The purines are adenine and guanine & pyrimidines are cytosine and uracil.

 Cahargaff’s rule-not obeyed: Due                Orcinol color reaction: RNAs can be histologically identified
 to the single—stranded nature, there is         by Orcinol color reaction due to the presence of ribose.
 no specific relation between purine and
 pyrimidine contents. Thus the guanine           Components:
 content is not equal to Cytosine (as is the     Nitrogenous bases: A,G, C, U
 case in DNA).                                   Sugar: Ribose sugar and H3PO4




                              Messenger RNA (mRNA)
          In 1961, the two Nobel laureates, Francois Jacob & Jacques Monad proposed the name messenger RNA
(mRNA). The mRNA carries the genetic information from DNA ribosomes (cytoplasm). The process of forming
mRNA on a DNA template is known as transcription. Although mRNAs formed from different genes can vary in
length but mRNS’s formed from a particular gene will have a different size.
          It is stable among all types of RNA. It has molecular weight of 2X106 and accounts for 5% of total RNA of
cell. Prokaryotic mRNA is metabolically unstable with high turnover rates & stable in eukaryotes with turn over rate
few hours to 24 hours. The mRNA is synthesized in the surface of DNA template. Thus it has been sequence
complimentary to DNA.
          The mRNA contains unique sequence of nucleotides. Each successive set of three nucleotides called
“Codon” (Provides the information for the ordered in corporation of amino acid in polypeptide during translation
process.
          In eukaryotic cells, a precursor heterogeneous nuclear RNA is first synthesized in the nucleoplasm, then it
is degraded by nuclear nucleus to mRNA which is then translocated to cytoplasm.Most of the eukaroyotic mRNA
are monocistronic i.e., they code only one polypeptide. In prokaryoteis mRNA are polycistronic i.e. they code for
more than one kind of polypeptide. The eukaryotic mRNA structure is composed of following four regions:
                                                      i) Introns
                                                      ii) Exons
                                                      iii) 5’-Methylated Cap
                                                      iv) 3’-poly A tail.
                           i. Introns: These are the Intervening sequences are non-coding regions. i.e., this sequence
                              does not have any protein code.
                          ii. Exons: these are the protein coding part. This is also called “Transcriptional Unit”.
                        iii. 5’-methylated cap: 7-methyl Guanosine is linked to the terminal 5’-phophate by enzyme
                              guanylate transferase through triphosphodiester linkage. Capping protects mRNA from
                              action of nucleases. It also stimulates translation process.
                         iv. 3’-poly A tail: The 3’-end of mRNA is terminated by long poly A chain consisting of
                              about 20 to 200 ribonucleotide residues. The poly A is synthesized by an enzyme with
                              from action of exonucleases. This tail region decides the life span of mRNA, which is
                              depends on the species.

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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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Functions of mRNA:
    •    The mRNA carries the genetic information from DNA to the protein biosynthetic machinery of ribosome.
    •    mRNA act as Xerox copy i.e., it carries the genetic information from DNA in Nucleolus to the rough
         Endoplasmic Reticulum (Site of Protein synthesis).




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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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                                    Ribosomal RNA (rRNA)
             •    This is the most stable form of RNA & is found in ribosomes.
             •    It has highest molecular weigh and sedimented during centrifugation at the speed of 1,000,000
                  rpm for 2 hours.
             •    rRNA is abundant of all tpes of RNAs. It makes of about 80% of the total RNA of cell.
             •    The ribosomes of prokaryotic & eukaryotic cells possess different species of RNA.
             •    rRNA has helical structure resulting from folding back of a single polymer at areas where
                  hydrogen bonding is possible.

                            Components                     Prokaryotic cell      Eukaryotic cell
                    1       Proteins                       35%                   50%
                    2       RNA                            65%                   50%
                    3       Sedimentation value            70s                   80s
                    4       Ribosomal subunits             30s & 50s             40s & 60s
                    5       rRNA’s                         16s (30s);            18s (40s);
                                                           23s & 5s (50s)        28s & 5s (60s)
                    6       Number of proteins             21 (30s)              34 (40s)
                                                           33 (50s)              50 (60s)

         Functions of Ribosomes:
                       Ribosomes are multicomponent
                  particles & contain several enzymatic        Svedberg Unit (S):
                  activities for protein synthesis.                     It is defined as “the velocity of sedimenting
                                                               molecule/unit gravitational field”. The ribosomal
                        •   Ribosomes bind to mRNA             RNA molecule care found in ribosomes. The 70s
                            in such a way that its codon       ribosomes of prokaryotes consists of 30s subunits &
                            can be matched to interact         50s subunit. The 30s subunit contains 16s rRNA
                            with anticodon of tRNA.            while the 50s subunit contains 23s & 5s rRNA
                            Thus rRNA binds mRNA &             molecules.
                            tRNA      with    ribosomes                The 80s ribosomes of eukaryoric consists of
                            during protein synthesis.          40s subunits 60s subunits. The 40s subunit contains
                                                               18s rRNA while the 60s contains 28s & 5s rRNA
                        •   Ribosomes provide site for         molecules.
                            binding        non-ribosomal
                            protein factors such as initiation factors, elongation factors & termination factors.

         Importance of rRNA:
                        •   The functions of the ribosomal RNA molecules in the ribosomal particle are not fully
                            understood, but they are necessary for ribosomal assembly and seem to play key roles in
                            the binding of mRNA to ribosomes and its translation.
                        •   Recent studies suggest that an rRNA component performs the peptidyl transferase
                            Activity and thus is an enzyme (a ribozyme).




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                                 Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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                                      Transfer RNA
                  Transfer RNA is the smallest form of RNA. It is also called “Soluble RNA”. The structure of
         tRNA (for alanine) was first elucidated by “Holley”. There are at least 20 species of tRNA molecules in
         every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein
         synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA
         molecules as a class have many features in common:

             •   It has molecular weight between
                 20,000 to 30,000.
              • It accounts for 15% of total
                 cellular RNA.
              • It contains 70 to 100 nucleotides.
                 tRNA carries amino acids to
                 mRNA during protein synthesis.
              • A prokaryotic cell contains 60
                 tRNA’s where as eukaryotic cell
                 contains 100 to 120 tRNAs. Out
                 of these 20 tRNA base sequence
                 had been determined.
              • Over 85% of tRNA has their 5’-
                 terminus bases Guanine while
                 the remainder cytosine.
              • All the tRNA have their 3’
                 terminus end sequence (-CCA),
                 where the amino acids are
                 charged. Each amino acid is
                 carried by a specific tRNA.
                 .
     The strucrure of tRNA, resembles that of a
clover leaf. tRNA contains mainly Four arms, each
with a base paired stem. All tRNA molecules
contain four main arms:
 1. Amino acid acceptor arm
 2. TψC arm
 3. Extra arm (or) Variable arm
 4. DHU arm
 5. Anticodon arm



1. Amino acid acceptor arm: This arm is capped with asequence CCA (5’ to 3’). The amino acid is attached to the
acceptor arm.

2. TψC arm: this arm contains a sequence of T, pseudouridine (represented by psi, ψ) and C.
3. Extra arm (or) Variable arm: This arm is the most variable in tRNA. Based on this variablilty, tRNA are
classified into 2 categories:
          a) Class I tRNA: The most predominant (about75%) form with 3-5 base pairs length.
          b) Class II tRNA: they contain 13 to 20 base pairs long arm.
4. DHU arm: It is so named due to the presence of dihydroxyuridine.
5. Anticodon arm: This arm, with the three specific nucleotide bases (anticodon), is resoponsible for the
recognition of triplet codon of mRNA. The codon and anticodon are complementary to each other.




                                                                                                                  16
                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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          THE CHEMICAL NATURE OF RNA DIFFERS FROM THAT OF DNA
         Ribonucleic acid (RNA) is a polymer of purine and pyrimidine ribonucleotides linked together by 3’, 5’-
phosphodiester bridges analogous to those in DNA. Although sharing many features with DNA, RNA possesses
several specific differences:

1. In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases are attached is ribose rather
than the 22-deoxyribose of DNA.

2. The pyrimidine components of RNA differ from those of DNA. Although RNA contains the ribonucleotides of
adenine, guanine, and cytosine, it does not possess thymine except in the rare case mentioned below. Instead of
thymine, RNA contains the ribonucleotide of uracil.

3. RNA exists as a single strand, whereas DNA exists as a double-stranded helical molecule. However, given the
proper complementary base sequence with opposite polarity, the single strand of RNA—as demonstrated, which is
capable of folding back on itself like a hairpin and thus acquiring double stranded Characteristics.

4. Since the RNA molecule is a single strand complementary to only one of the two strands of a gene, its guanine
content does not necessarily equal its cytosine content, nor does its adenine content necessarily equal its uracil
content.



                                 Differences between RNA and DNA

                                          RNA                                             DNA
Location                  Cytoplasm                                    Nucleus
Number of Base            Usually 100 to 5000 base                     Million of base pairs Double stranded
Strands                   Generally Single stranded                    Double stranded
Sugar moiety              Ribose                                       Deoxyribose
Nitrogenous base          Adenine, Guanine                             Adenine, Guanine
                          Cytosine, Thymine                            Cytosine, Uracil
Chargaff’s rule           Guanine content is not equal to              Guanine content is equal to cytosine
                          cytosine and adenine is not equal            and adenine is equal to Uracil
                          to Uracil
Alkali treatment          Easily destroyed by alkali                   Alkali resistant




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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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               Sequencing of Nucleic Acids
          Two basic protocols for nucleic acid sequencing are in widespread use: the chain termination or dideoxy
method of F. Sanger and the base-specific chemical cleavage method developed by A. M. Maxam and W. Gilbert.
Because both methods are carried out on nanogram amounts of DNA, very sensitive analytical techniques are used
to detect the DNA chains following electrophoretic separation on polyacrylamide gels.
         Typically, the DNA molecules are labeled with radioactive 32P,1 and following electrophoresis, the pattern
of their separation is visualized by autoradiography. A piece of X-ray film is placed over the gel and the
radioactive disintegrations emanating from 32P decay create a pattern on the film that is an accurate image of the
resolved oligonucleotides. Recently, sensitive biochemical and chemiluminescent methods have begun to supersede
the use of radioisotopes as tracers in these experiments.

1. Chain Termination (or) Dideoxy Method(or) Sanger’s dideoxy Method:
           To appreciate the rationale of the chain termination or dideoxy method, we first must briefly examine the
biochemistry of DNA replication. DNA is a double- helical molecule. In the course of its replication, the sequence
of nucleotides in one strand is copied in a complementary fashion to form a new second strand by the enzyme DNA
polymerase.
           Each original strand of the double helix serves as template for the biosynthesis that yields two daughter
DNA duplexes from the parental double helix. DNA polymerase carries out this reaction in vitro in the presence of
the four deoxynucleotide monomers and copies single-stranded DNA, provided a double-stranded region of DNA is
artificially generated by adding a primer.
           This primer is merely an oligonucleotide capable of forming a short stretch of dsDNA by base pairing with
the ssDNA. The primer must have a free 3’-OH end from which the new polynucleotide chain can grow as the first
residue is added in the initial step of the polymerization process. DNA polymerases synthesize new strands by
adding successive nucleotides in the 5’ 3’ direction.

Chain Termination Protocol
         In the chain termination method of DNA sequencing, a DNA fragment of unknown sequence serves as
template in a polymerization reaction using some type of DNA polymerase, usually Sequenase, and a genetically
engineered version of bacteriophage T7 polymerase that lacks all traces of exonuclease activity that might otherwise
degrade the DNA.
         The primer requirement is met by an appropriate oligonucleotide (this method is also known as the primed
synthesis method for this reason). Four parallel reactions are run; all four contain the four deoxynucleoside
triphosphates dATP, dGTP, dCTP, and dTTP, which are the substrates for DNA polymerase. In each of the four
reactions, a different 2’, and 3’-dideoxynucleotide is included; and it is these dideoxynucleotides that give the
method its name. Because dideoxynucleotides lack 3’-OH groups, these nucleotides cannot serve as acceptors for 5’-
nucleotide addition in the polymerization reaction, and thus the chain is terminated where they become incorporated.
         The concentrations of the four deoxynucleotides and the single dideoxynucleotide in each reaction mixture
are adjusted so that the dideoxynucleotide is incorporated infrequently. Therefore, base-specific premature chain
termination is only a random, occasional event, and a population of new strands of varying length is synthesized.
Four reactions are run, one for each dideoxynucleotide, so that termination, although random, can occur everywhere
in the sequence. In each mixture, each newly synthesized strand has a dideoxynucleotide at its 3’-end, and its
presence at that position demonstrates that a base of that particular kind was specified by the template. A
radioactively labeled dNTP is included in each reaction mixture to provide a tracer for the products of the
polymerization process.

Reading Dideoxy Sequencing Gels
         The sequencing products are visualized by autoradiography (or similar means) following their separation
according to size by polyacrylamide gel electrophoresis. Because the smallest fragments migrate fastest upon
electrophoresis and because fragments differing by only single nucleotides in length are readily resolved, the
autoradiogram of the gel can be read from bottom to top, noting which lane has the next largest band at each step.
Thus, the gel is read AGCGTAGC (5’ 3’). Because of the way DNA polymerase acts, this observed sequence is
complementary to the corresponding unknown template sequence. Knowing this, the template sequence now can be
written GCTACGCT (5’ 3’).

                                                                                                                 18
                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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Life science Study materials                                              Chemistry of Nucleic Acids
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2. Base-Specific Chemical Cleavage Method (or) Maxam-Gilbert Method:
         The base-specific chemical cleavage (or Maxam–Gilbert) method starts with a single-stranded
DNA that is labeled at one end with radioactive 32P. (Doublestranded DNA can be used if only one strand
is labeled at only one of its ends.) The DNA strand is then randomly cleaved by reactions that specifically
fragment its sugar–phosphate backbone only where certain bases have been chemically removed. There is
no unique reaction for each of the four bases.

         However, there is a reaction specific to G only and a purine-specific reaction that removes A or
G. Thus, the difference in these two reactions is a specific indication of where A occurs. Similarly, there
is a cleavage reaction specific for the pyrimidines (C+T), which, if run in the presence of 1 or 2 M NaCl,
works only with C. Differences in these two are thus attributable to the presence of T in the nucleotide
sequence. Note that the key to Maxam–Gilbert sequencing is to modify a base chemically so that it is
removed from its sugar.

         Then piperidine excises the sugar from its 5’- and 3’-links in a β-elimination reaction. The
conditions of chemical cleavage described in a single scission occurs per DNA molecule. However,
because a very large number of DNA molecules exist in each reaction mixture, the products are a random
collection of different-sized fragments wherein the occurrence of any base is represented by its unique
pair of 5’- and 3’-cleavage products.

         These products form a complete set, the members of which differ in length by only one
nucleotide, and they can be resolved by gel electrophoresis into a “ladder,” which can be visualized by
autoradiography of the gel if the DNA fragments are radioactively labeled. In principle, the Maxum–
Gilbert method can provide the total sequence of a dsDNA molecule just by determining the purine
positions on one strand and then the purines on the complementary strand. Complementary base-pairing
rules then reveal the pyrimidines along each strand, T complementary to where A is, C complementary to
where G occurs. (The analogous approach of locating the pyrimidines on each strand would also provide
sufficient information to write the total sequence.)

         With current technology, it is possible to read the order of as many as 400 bases from the
autoradiogram of a sequencing gel. The actual chemical or enzymatic reactions, electrophoresis, and
autoradiography are now routine, and a skilled technician can sequence about 1 kbp per week using these
manual techniques. The major effort in DNA sequencing is in the isolation and preparation of fragments
of interest, such as cloned genes.




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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
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                                                                                                               21
                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
------------------------------------------------------------------------------------------------------------
3. Automated DNA Sequencing
         In recent years, automated DNA sequencing machines capable of identifying about 104 bases per day have
become commercially available. One clever innovation has been the use of fluorescent dyes of different colors to
uniquely label the primer DNA introduced into the four sequencing reactions; for example, red for the A reaction,
blue for T, green for G, and yellow for C.

        Then, all four reaction mixtures can be combined and run together on one electrophoretic gel slab. As the
oligonucleotides are separated and pass to the bottom of the gel, each is illuminated by a low-power argon laser
beam that causes the dye attached to the primer to fluoresce.

          The color of the fluorescence is detected automatically, revealing the identity of the primer, and hence the
base, immediately. The development of such automation has opened the possibility for sequencing the entire human
genome, some 2.9 billion bp. Even so, if 100 automated machines operating at peak efficiency were dedicated to
the task, it would still take at least 8 years to complete!




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                                Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
------------------------------------------------------------------------------------------------------------

Reference Books:

    1. Principles’ of Biochemistry, Lehninger, Nelson & Cox, 3/e, worth publications

    2. Harper’s Illustrated Biochemistry, Murray, Lange Publications

    3. Text book of Biochemistry, 5/e, Vasudevan, Jaypee publications

    4. Text book of Biochemistry West & Todd, Oxford Publications

    5. Fundamentals of Biochemistry, J.L.Jain, S.Chand’s Publications

    6. Medical Biochemistry, Chatarjee & Shindae, Jaypee Publications




                                      Why I am a Biologist
People some times ask me why I am a biologist. After much serious thought, I came up with this explanation:

When I first started out, I was going to be a mathematician. So I took algebra, but I was going to be a
mathematician. So I took algebra, but I found that was highly variable.

So, I tried Geometry and that’s were I learned all the angles. Then I took calculus. That was truly an integrating
experience, but it definitely had its limits. After a great deal of consideration, I decided to turn away from math and
give some serious thought to science.

I tried Geology, but found that was kind of hard.

Next I tried Physics but I knew that would never work.

And even though I had heard chemists had all the solutions,

I finally opted for Biology because, after all, it’s a   living.


                                                                   (Most of the biologist’s opinion)




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                                   Prepared by I.Satish Kumar, Biochemistry Lecturer
Life science Study materials                                              Chemistry of Nucleic Acids
------------------------------------------------------------------------------------------------------------

                                CSIR means Council of Scientific Industrial and Research.
                                                     CSIR Laboratories
Institute of Genomics and Integrative Biology                             Indian Institute of Chemical Biology
Near Jubilee Hall, Delhi University Campus, Mall Road, Delhi 110          4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032, West
007                                                                       Bengal
Telephone: 011-27666156/6157/7602/7439                                    Telephone: 033-24730492/ 3492
Fax: 011-27667471                                                         Fax: 033-24730286
Email: info@igib.res.in                                                   Email: (Director): director@iicb.res.in
Web: http://www.igib.res.in/                                              Web: http://www.iicb.res.in/
Research Areas: Allergy and immunology, diagnostics, genetic              Research Areas: Natural products of medicinal, biological and
engineering, bio-organics and high-tech reagents.                         industrial value, development of innovative immunoassay
                                                                          techniques, development of tissue-targeted drug-delivery system.

Centre for Cellular & Molecular Biology
Uppal Road, Hyderabad 500 007, Andhra Pradesh                             Indian Institute of Chemical Technology
Telephone: 040-27160222-41                                                Uppal Road, Hyderabad 500 007, Andhra Pradesh
Fax: 040-27160591/ 0311                                                   Telephone: 040-27160123
Email: (Director): lalji@ccmb.res.in                                      Fax: 040-27160387
Web: http://www.ccmb.res.in/                                              Email: kvr@iict.ap.nic.in, sampath@iict.ap.nic.in
Research Areas: Biophysics & biochemistry, molecular biology,             Web: http://www.iictindia.org/
genetics & evolution, biomedicines & biotechnology.                       Research Areas: Development of technologies for pesticides,
                                                                          drugs, organic intermediates and fine chemicals.

Central Drug Research Institute
Chattar Manzil Palace, Post Box No. 173, Lucknow 226 001, Uttar           Institute of Microbial Technology
Pradesh                                                                   Sector 39-A, Chandigarh 160 036
Telephone: 0522-2212411-18, 2212439                                       Telephone: 0172-695225/226/219, 690025/173/908
Fax: 0522-2223405, 2223938                                                Fax: 0172-690585/ 632
Email: info@cdriindia.org                                                 Email: raghava@imtech.res.in
Web: http://www.cdriindia.org/                                            Web: http://www.imtech.res.in/
Research Areas: Development of contraceptives, new drugs for              Research Areas: Molecular biology and microbial genetics,
tropical diseases (malaria, filariasis, leishmaniasis), cardio-vascular   animal cell/tissue culture and protein engineering.
and central nervous system disorders.

                                                                          National Botanical Research Institute
Central Food Technological Research Institute                             Rana Pratap Marg, Lucknow 226 001, Uttar Pradesh
Mysore 570 013, Karnataka                                                 Telephone: 0522-2205848
Telephone: 0821-2515910/ 2514760                                          Fax: 0522-2205839
Fax: 0821-2517233                                                         Email: p.pushpangadan@nbri.res.in
Email: prp@cscftri.ren.nic.in                                             Web: http://www.nbri-lko.org/
Web: http://www.cftri.com/                                                Research Areas: Plant biotechnology, environmental sciences,
Research Areas: Development of food products and processes for            taxonomy and ethnobotany, plant molecular biology.
optimal utilization of country's agricultural produce, upgrading
traditional food technology & development of appropriate
technologies for reducing and eliminating post-harvest losses of          National Chemical Laboratory
perishables and durables, bioactive substances and food packaging         Dr. Homi Bhabha Road, Pune 411 008, Maharashtra
                                                                          Telephone: 020-20-5893030
                                                                          Fax: 020-5893355
Central Institute of Medicinal and Aromatic Plants                        Email: director@ems.ncl.res.in
P.O. CIMAP, Near Kukrail Picnic Spot, Lucknow 226 015, Uttar              Web: http://www.ncl-india.org/
Pradesh                                                                   Research Areas: Catalysis, biotechnology, organic chemical
Telephone: 0522-2359623                                                   technology, basic research in chemistry and biochemistry.
Fax: 0522-2342666
Email: director@cimap.res.in
                                                                          Institute of Genomics and Integrative Biology
Web: http://www.cimap.res.in/
                                                                          Near Jubilee Hall, Delhi University Campus, Mall Road, Delhi 110
Research Areas: Development of agrotechnologies for
                                                                          007
economically important medicinal and aromatic plants, basic
                                                                          Telephone: 011-27666156/6157/7602/7439
research in the area of phytochemistry,
                                                                          Fax: 011-27667471
plant physiology and biochemistry, pathology, genetics,
                                                                          Email: info@igib.res.in
entomology and pharmacognosy
                                                                          Web: http://www.igib.res.in/
                                                                          Research Areas: Allergy and immunology, diagnostics, genetic
                                                                          engineering, bio-organics and high-tech reagents.




                                                                                                                                         24
                                          Prepared by I.Satish Kumar, Biochemistry Lecturer