Nucleic Acids

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• Nucleic Acids

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Nucleic Acids Components

• Nucleic acid A biopolymer containing three types
  of monomer units.
• Heterocyclic aromatic amine bases derived from
  purine and pyrimidine.
• The monosaccharides D-ribose or 2-deoxy-D-ribose
• Phosphoric acid.

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Purine/Pyrimidine Bases
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Nucleosides Definition

• Nucleoside A building block consisting of
• D-ribose or 2-deoxy-D-ribose
• heterocyclic aromatic amine base
• b-N-glycosidic bond .

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Nucleotides Definition

• Nucleotide Phosphoric acid ester of a
  nucleoside, most commonly either the 3 or the 5
  OH.

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Nucleotides

• Example Identify these nucleotides.

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DNA - 1 Structure

• Deoxyribonucleic acids (DNA)
• A backbone of alternating units of
  2-deoxy-D-ribose and phosphate in which the 3-OH
  of one 2-deoxy-D-ribose is joined by a
  phosphodiester bond to the 5-OH of another
  2-deoxy-D-ribose.
• Primary Structure The sequence of bases along
  the pentose-phosphodiester backbone of a DNA
  molecule (or an RNA molecule).
• Read from the 5 end to the 3 end.

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DNA - 1 Structure

• A structural formula for TG phosphorylated at the
  5 end.

Thymine, T
Guanine, G
3
diester
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DNA - 2 Structure

• Secondary structure The ordered arrangement of
  nucleic acid strands.
• The double helix model of DNA 2 structure was
  proposed by James Watson and Francis Crick in
  1953.
• Double helix A type of 2 structure of DNA
  molecules in which two antiparallel
  polynucleotide strands are coiled in a
  right-handed manner about the same axis.

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DNA - 2 Structure

• Hydrogen bonding occurs between bases
• A---T
• G---C
• Evidence Base composition in mole-percent of DNA
  for several organisms.

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Base Pairing

• Base-pairing between adenine and thymine (A-T)
  and guanine and cytosine (G-C).

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Double Helix

• Ribbon model of double-stranded B-DNA.

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Forms of DNA

• B-DNA
• the predominant form in dilute aqueous solution.
• a right-handed helix.
• 2000 pm thick with 3400 pm per ten base pairs.
• minor groove of 1200pm and major groove of 2200
  pm.
• A-DNA
• a right-handed helix, but thicker than B-DNA.
• 2900 pm per 10 base pairs.
• Z-DNA
• a left-handed double helix.

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Double Helix

• An idealized model of B-DNA.

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DNA - 3 Structure

• Tertiary structure The three-dimensional
  arrangement of all atoms of a double-stranded
  DNA, commonly referred as supercoiling.
• Circular DNA A type of double-stranded DNA in
  which the 5 and 3 ends of each stand are joined
  by phosphodiester bonds.
• Histone A protein, particularly rich in the
  basic amino acids lysine and arginine, that is
  found associated with DNA molecules.

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DNA - 3 Structure

• Figure 28.10 Relaxed and supercoiled DNA.

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DNA - 3 Structure

• Chromatin Consists of DNA molecules wound around
  particles of histones (a simple protein
  containing mainly basic amino acids) in a
  beadlike structure.
• Further coiling produces the dense chromatin
  found in nuclei of plant and animal cells.

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Ribonucleic Acids (RNA)

• RNA
• long, unbranched chains of nucleotides joined by
  phosphodiester groups between the 3-OH of one
  pentose and the 5-OH of the next
• Consists of A, U ( Uracil), G, C.
• the pentose unit in RNA is ?-D-ribose rather than
  ?-2-deoxy-D-ribose.
• the pyrimidine bases in RNA are uracil and
  cytosine rather than thymine and cytosine.
• RNA is single stranded rather than double
  stranded.

(RNA)
A Uracil
C Cytosine
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rRNA

• Different types of RNA
• Ribosomal RNA (rRNA) A ribonucleic acid found in
  ribosomes, the site of protein synthesis.

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tRNA

• Transfer RNA (tRNA) A ribonucleic acid that
  carries a specific amino acid to the site of
  protein synthesis on ribosomes.

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mRNA

• Messenger RNA (mRNA) A ribonucleic acid that
  carries coded genetic information from DNA to the
  ribosomes for the synthesis of proteins.
• Present in cells in relatively small amounts and
  very short-lived.
• Single stranded.
• mRNA synthesis is directed by information encoded
  on DNA.
• A complementary strand of mRNA is synthesized
  along one strand of an unwound DNA, starting from
  the 3 end.

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mRNA from DNA, transcription
DNA (RNA)
A T(U)
G C(C)
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The Genetic Code
second
first
third
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The Genetic Code

• Properties of the Code
• Only 61 triplets code for amino acids the
  remaining 3 (UAA, UAG, and UGA) signal chain
  termination.
• The code is degenerate, which means that several
  amino acids are coded for by more than one
  triplet. Leu, Ser, and Arg, for example, are
  each coded for by six triplets.
• Degenerate triplets differ only in the third
  letter of the codon that varies. Gly, for
  example, is coded for by GGA, GGG, GGC, and GGU.
  (GG? Codes for GLY)
• There is no ambiguity in the code each triplet
  codes for one and only one amino acid.

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Sequencing DNA

• Restriction endonuclease An enzyme that
  catalyzes hydrolysis of a particular
  phosphodiester bond within a DNA strand.
• Over 1000 endonucleases have been isolated and
  their specificities determined.
• Typically they recognize a set sequence of
  nucleotides and cleave the DNA at or near that
  particular sequence.
• EcoRI (eco R 1) from E. coli, for example,
  cleaves as shown.

Recognition pattern
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Sequencing DNA

• Following are several more examples of
  endonucleases and their specificities.

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Sequencing DNA

• Maxam-Gilbert method A method developed by Allan
  Maxam and Walter Gilbert depends on
  base-specific chemical cleavage.
• Dideoxy chain termination method Developed by
  Frederick Sanger.
• Gilbert and Sanger shared the 1980 Nobel Prize
  for biochemistry for their development of
  chemical and biochemical analysis of DNA
  structure.

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Replication in Vitro

• the sequence of nucleotides in one strand (ssDNA)
  is copied as a complementary strand to form the
  second strand of a double-stranded DNA (dsDNA).
• Synthesis is catalyzed by DNA polymerase.
• DNA polymerase requires
• the four deoxynucleotide triphosphate (dNTP)
  monomers
• a primer is present to start the process.

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Dideoxy Chain Termination

• Chain termination method is accomplished by the
  addition to the synthesizing medium of a
  2,3-dideoxynucleotide triphosphate (ddNTP).
• Because a ddNTP has no 3-OH, chain synthesis is
  terminated when a ddNTP becomes incorporated.

Without a OH here chain cannot extend.
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Methodology of Dideoxy Chain Termination

• In this method, the following are mixed
• Single-stranded DNA of unknown sequence and
  primer then divided into four reaction mixtures.
• To each of the four reaction mixture is then
  added
• The four dNTP, one of which is labeled in the 5
  end with phosphorus-32 which is radioactive.
• DNA polymerase.
• one of the four ddNTPs.

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Dideoxy Chain Termination

• After gel electrophoresis of each reaction
  mixture
• a piece of film is placed over the gel.
• Gamma rays released by P-32 darken the film and
  create a pattern of the resolved oligonucleotide.
• The base sequence of the complement to the
  original strand is read directly from bottom to
  top of the developed film.

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Dideoxy Chain Termination

• The primer-DNA template is divided into four
  separate reaction mixtures. To each is added the
  four dNTPs, DNA polymerase, primer and one of the
  four ddNTPs in small amounts. Synthesis will
  produce chains of varying lengths.

DNA
A T
G C
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Dideoxy Chain Termination

• The mixtures are separated by polyacrylamide gel
  electrophoresis.

From the four different ddNTP reaction mixtures.
Move from 5 to 3 end.