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Nucleic AcidsNucleic Acids

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Nucleic Acids ComponentsNucleic Acids Components

Nucleic acid: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 BasesPurine/Pyrimidine Bases

HN

N

O

O

H

N

N

NH2

O

H

HN

N

O

O

H

CH3N

N

HN

N N

N

O

HH2N

N

N N

N

NH2

H

N

N N

N

H

Uracil (U) Thymine (T) Cytosine (C)Pyrimidine

1

2

34

5

6

Guanine (G)Adenine (A)Purine

1

2

34

56 7

8

9

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NucleoNucleossides Definitionides Definition

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

HH

HOHOCH2

HO OH

O

O

HN

N

H anomericcarbon

a -N-glycosidicbond

Uridine

-D-riboside

uracil

1'

2'3'

4'

5'

1

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NucleoNucleottides Definitionides Definition

NucleoNucleottide:ide: Phosphoric acid ester of a nucleoside, most commonly either the 3’ or the 5’ OH.

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OP

O-

O-O

5'

Adenosine 5'-monophosphate(AMP)

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Acyclovir & AZTAcyclovir & AZT

Azidothymidine (AZT) Acyclovir (drawn to show itsstructural relationship to 2-deoxyguanosine

O

O

N

HN

HOC

HN3

H

H

HH

O

CH3

O

H HHH

HOC

H2N

HN

N

O

N

N

H2H2

Used to treat HIV

It is used to treat or prevent infections caused by certain kinds of viruses. Examples of these infections include herpes and shingles

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DNA - 1° StructureDNA - 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: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° StructureDNA - 1° Structure

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

NO

HN

-O-P-O-CH2

O

HH

OH

HH

H

OCH3

O=P O CH2

O

-O

O-

NH

NN

N

O

NH2

O

HH

O

HH

H

5'

3'

phosphorylated5' end

free 3' end

Thymine, T

Guanine, G3

diester

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DNA - 2° StructureDNA - 2° Structure

Secondary 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: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° StructureDNA - 2° Structure

• Hydrogen bonding occurs between bases• A---T

• G---C

• Evidence: Base composition in mole-percent of DNA for several organisms.

A C TOrganism G A/T G/CPurines/Pyrimidines

HumanSheepYeast

E. coli

30.4 19.9 19.9 30.1 1.01 1.00 1.0129.3 21.4 21.0 28.3 1.04 1.02 1.0331.7 18.3 17.4 32.6 0.97 1.05 1.0026.0 24.9 25.2 23.9 1.09 0.99 1.04

Purines Pyrimidines

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Base PairingBase Pairing

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

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Double HelixDouble Helix

• Ribbon model of double-stranded B-DNA.

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Forms of DNAForms 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 HelixDouble Helix

• An idealized model of B-DNA.

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DNA - 3° StructureDNA - 3° Structure

Tertiary structure:Tertiary structure: The three-dimensional arrangement of all atoms of a double-stranded DNA, commonly referred as supercoiling.

Circular DNA:Circular DNA: A type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds.

Histone: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° StructureDNA - 3° Structure

Chromatin: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)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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rRNArRNA

Different types of RNA:

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

Molecular WeightRange (g/mol)

Number ofNucleotides

Percentageof Cell RNA

mRNA 25,000 - 1,000,000 75 - 3,000 2tRNA 23,000 - 30,000 73 - 94 16rRNA 35,000 - 1,100,000 120 - 2904 82

Type

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tRNAtRNA

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

OBase

OHO

CH

RNH3

+

tRNA-O-P-O-CH2

amino acid, boundas an ester to itsspecific tRNA

HH H

H

C=O

O

O-

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mRNAmRNA

Messenger RNA (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, transcriptionmRNA from DNA, transcription

DNA (RNA)

A – T(U)

G – C(C)

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UUU PheUUC PheUUA LeuUUG Leu

UCU SerUCC SerUCA SerUCG Ser

UAU TyrUAC TyrUAA StopUAG Stop

UGU CysUGC CysUGA StopUGG Trp

GUCAG

CUU LeuCUC LeuCUA LeuCUG Leu

CCU ProCCC ProCCA ProCCG Pro

CAU HisCAC HisCAA GlnCAG Gln

CGU ArgCGC ArgCGA ArgCGG Arg

UCAG

G

AUU IleAUC IleAUA IleAUG* Met

ACU ThrACC ThrACA ThrACG Thr

AAU AsnAAC AsnAAA LysAAG Lys

AGU SerAGC SerAGA ArgAGG Arg

UCAG

UCAG

GUU ValGUC ValGUA ValGUG Val

GCU AlaGCC AlaGCA AlaGCG Ala

GAU AspGAC AspGAA GluGAG Glu

GGU GlyGGC GlyGGA GlyGGG Gly

*AUG also serves as the principal initiation codon.

U

U C A

C

A

The Genetic CodeThe Genetic Code

first

second

third

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The Genetic CodeThe 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 DNASequencing DNA

Restriction endonuclease: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.

5' G-A-A-T-T-C---3'EcoRI

5' G + 5'-A-A-T-T-C---3'

cleavage here

Recognition pattern

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Sequencing DNASequencing DNA

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

AluI AG CT

BalI TGG CCA

FnuDII CG CG

HeaIII GG CC

Not I GC GGCCGC

Mbol GATC

SacI GAGCT C

Enzyme EnzymeRecognitionSequence

RecognitionSequence

HpaII C CGG

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Sequencing DNASequencing DNA

• Maxam-Gilbert method:Maxam-Gilbert method: A method developed by Allan Maxam and Walter Gilbert; depends on base-specific chemical cleavage.

• Dideoxy chain termination methodDideoxy 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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ReplicationReplication in Vitro 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 TerminationDideoxy 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.

-O-P-O-P-O-P-O-CH2

O-

O

O- O-

O

H

Base

H H

H HO

H

A 2',3'-dideoxynucleoside triphosphate (ddNTP)

O

Without a OH here chain cannot extend.

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MethodologyMethodology of Dideoxy Chain Termination 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 TerminationDideoxy 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 TerminationDideoxy 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 TerminationDideoxy Chain Termination

The mixtures are separated by polyacrylamide gel electrophoresis. From the four different ddNTP reaction mixtures.

Move from 5’ to 3’ end.