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DNA STRUCTUREDNA STRUCTURE
NUCLEIC ACIDSNUCLEIC ACIDS
Nucleic acids are polymersNucleic acids are polymers Monomer---nucleotidesMonomer---nucleotides
Nitrogenous basesNitrogenous bases PurinesPurines PyrimidinesPyrimidines
Sugar Sugar RiboseRibose DeoxyriboseDeoxyribose
PhosphatesPhosphates +nucleoside=nucleotide+nucleoside=nucleotide
}Nucleosides
The Sugars The Sugars
The Bases The Bases
PURINES
PYRIMIDINES
Bases of DNA (and RNA)Bases of DNA (and RNA)Bases of DNA (and RNA)Bases of DNA (and RNA)
RNA only DNA only
Purines:
Pyrimidines:
Nucleotides and Nucleotides and NucleosidesNucleosides
Chemical Structure of DNA and RNA
Chemical Structure of DNA and RNA
Figure 4.1
RNA DNA
Nucleotide
Nucleoside
1’
2’
4’
The C is named 1’-5’
Resume
Nucleotides and Nucleotides and NucleosidesNucleosides
BASEBASE NUCLEOSIDENUCLEOSIDE DEOXYNUCLEOSIDEOXYNUCLEOSIDEDE
AdenineAdenine AdenosineAdenosine 2-2-deoxyadenosinedeoxyadenosine
GuanineGuanine GuanosineGuanosine 2-2-deoxyguanosinedeoxyguanosine
CytosineCytosine CytodineCytodine 2-deoxycytodine2-deoxycytodine
UracilUracil UridineUridine Not usually found Not usually found
ThymineThymine Not usually foundNot usually found 2-deoxythymidine2-deoxythymidine
Nucleotides are nucleosides + phosphate
NucleotidNucleotide e
Analogs Analogs as Drugsas Drugs
make up 13-34% of the dry weight in bacteriadeoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
O
C C
C C
Base
H
H or OH
H H
H
H
CH2OP
OH
O
HO
Nucleotide: a building block
Sugar:• RNA – ribose (OH)• DNA – deoxyribose (H)
Bases:• adenine (A), cytosine (C), guanine (G), thymine (T)• RNA uses uracil (U) instead of thymine
Nucleoside: base + sugar
• certain nucleotides serve as a storage of energy and reducing powere.g. ATP -> ADP -> AMP
hydrolysis (energy is released)
Nucleic Acids
DNA Stabilization– DNA Stabilization– Complementary Base PairingComplementary Base Pairing
DNA Stabilization-Base DNA Stabilization-Base StackingStacking
DNA Stabilization--H-bonding DNA Stabilization--H-bonding between DNA base pair stacks between DNA base pair stacks
Advantages to Double HelixAdvantages to Double Helix
Stability---protects bases from attack Stability---protects bases from attack by Hby H22O soluble compounds and HO soluble compounds and H22O O itself.itself.
Provides easy mechanism for Provides easy mechanism for replicationreplication
Physical Structure (cont’d)Physical Structure (cont’d)
Chains are anti-parallel (i.e in opposite Chains are anti-parallel (i.e in opposite directions)directions)
Diameter and periodicity are Diameter and periodicity are consistent consistent 2.0 nm2.0 nm 10 bases/ turn10 bases/ turn 3.4 nm/ turn3.4 nm/ turn
Width consistent because of Width consistent because of pyrimidine/purine pairingpyrimidine/purine pairing
Physical Structure (cont’d)Physical Structure (cont’d)
G-C ContentG-C Content
A=T, G=C, but ATA=T, G=C, but AT≠GC≠GC Generally GC~50%, but extremely Generally GC~50%, but extremely
variablevariable EX.EX.
Slime mold~22%Slime mold~22% Mycobacterium~73%Mycobacterium~73%
Distribution of GC is not uniform in Distribution of GC is not uniform in genomesgenomes
CONSEQUENCES OF GC CONSEQUENCES OF GC CONTENTCONTENT
GC slightly denser GC slightly denser Higher GC DNA moves further in a Higher GC DNA moves further in a
gradientgradient Higher # of base pairs=more stable Higher # of base pairs=more stable
DNA, i.e. the strands don’t separate DNA, i.e. the strands don’t separate as easily. as easily.
FORMS OF DNAFORMS OF DNA
Supercoiling Supercoiling
Cruciform Cruciform StructuresStructures
Another adaptation to supercoilingAssociated with palindromes
DNA is DynamicDNA is Dynamic Like proteins, DNA has 3Like proteins, DNA has 3ºº structure structure Why so many deviations from normal Why so many deviations from normal
conformation? conformation? Effects on transcription (gene Effects on transcription (gene
expression)expression) Enhances responsivenessEnhances responsiveness May also serve in packagingMay also serve in packaging
NOTE: most cellular DNA exists as NOTE: most cellular DNA exists as protein containing supercoilsprotein containing supercoils
Denaturation of DNADenaturation of DNA Denaturation by Denaturation by
heating.heating. How observed?How observed?
AA260260 For dsDNA, For dsDNA,
AA260260=1.0 for 50 =1.0 for 50 µg/mlµg/ml For ssDNA and RNA For ssDNA and RNA
AA260260=1.0 for 38 =1.0 for 38 µg/mlµg/ml For ss oligosFor ss oligos
AA260260=1.0 for 33 =1.0 for 33 µg/mlµg/ml Hyperchromic shiftHyperchromic shift
The T at which ½ the DNA sample is denatured is called the melting temperature (Tm)
Importance of TImportance of Tmm
Critical importance in any technique Critical importance in any technique that relies on complementary base that relies on complementary base pairing pairing Designing PCR primersDesigning PCR primers Southern blotsSouthern blots Northern blotsNorthern blots Colony hybridization Colony hybridization
Factors Affecting TFactors Affecting Tmm
G-C content of sampleG-C content of sample Presence of intercalating agents Presence of intercalating agents
(anything that disrupts H-bonds or (anything that disrupts H-bonds or base stacking)base stacking)
Salt concentrationSalt concentration pH pH LengthLength
RenaturationRenaturation
Strands can be induced to renature Strands can be induced to renature (anneal) under proper conditions. (anneal) under proper conditions. Factors to consider:Factors to consider: TemperatureTemperature Salt concentrationSalt concentration DNA concentrationDNA concentration TimeTime
CCoot Curvest Curves
What Do CWhat Do Coot Curves Reveal?t Curves Reveal?
Complexity of DNA sampleComplexity of DNA sample Reveals important info about the Reveals important info about the
physical structure of DNAphysical structure of DNA Can be used to determine TCan be used to determine Tmm for for
techniques that complementary base techniques that complementary base pairing. pairing.
Complexity of DNA- FactorsComplexity of DNA- FactorsRepetitive Sequences Repetitive Sequences
Single Copy GenesSingle Copy Genes Highly repetitive (hundreds to millions)Highly repetitive (hundreds to millions)
Randomly dispersed or in tandem repeatsRandomly dispersed or in tandem repeats Satellite DNASatellite DNA
Microsatellite repeatsMicrosatellite repeats Miniisatellite repeatsMiniisatellite repeats
Middle repetitive (10- hundreds)Middle repetitive (10- hundreds) ClusteredClustered DispersedDispersed
Slightly repetitive (2-10 copies)Slightly repetitive (2-10 copies)
Highly repetitive sequences
Middle repetitiveMiddle repetitive sequences
Unique sequences
Renaturation curves of E. coli and calf DNA
RNA RNA
TypesTypes mRNAmRNA tRNAtRNA rRNArRNA
It’s still an RNA worldIt’s still an RNA world snRNAsnRNA siRNAsiRNA RibozymesRibozymes
Behavior in AcidsBehavior in Acids Dilute or mild acidic conditionsDilute or mild acidic conditions Intermediate conditions. EX. 1N HCl Intermediate conditions. EX. 1N HCl
@ 100@ 100ºC for 15m : DepurinationºC for 15m : Depurination Harsher treatment-EX. 2-6N HCl, Harsher treatment-EX. 2-6N HCl,
higher temps: Depyrimidination.higher temps: Depyrimidination. NOTE: some phosphodiester bond NOTE: some phosphodiester bond
cleavage observed during cleavage observed during depurination, much more during depurination, much more during depyrimidinationdepyrimidination
Behavior in BasesBehavior in Bases N-glycosidic bonds stable in mild N-glycosidic bonds stable in mild
alkaline conditionsalkaline conditions DNA meltsDNA melts PhosphodiesterPhosphodiester linkages in DNA and linkages in DNA and
RNA show very different behavior in RNA show very different behavior in weak bases (EX 0.3 N KOH @37weak bases (EX 0.3 N KOH @37ººC ~1 C ~1 hr.)hr.)
RNA Hydrolysis in Alkaline RNA Hydrolysis in Alkaline SolutionsSolutions
2,3 cyclic nucleotide
Hydrolysis by EnzymesHydrolysis by Enzymes
Nuclease—catalyzes hydrolysis of Nuclease—catalyzes hydrolysis of phosphodiester backbone phosphodiester backbone ExonucleasesExonucleases EndonucleasesEndonucleases
General. Ex DNAse IGeneral. Ex DNAse ISpecific Ex. Restriction Specific Ex. Restriction endonucleasesendonucleases
RibozymesRibozymes
Restriction Restriction EnzymesEnzymes
RIBOZYMERIBOZYMESS
Catalytic Catalytic RNARNA
Can work Can work alone or with alone or with proteinsproteins
Therapeutic Therapeutic applications?applications?
SEQUENCINGSEQUENCING
Purpose—determine nucleotide Purpose—determine nucleotide sequence of DNA sequence of DNA
Two main methodsTwo main methods Maxam & Gilbert, using chemical
sequencing Sanger, using dideoxynucleotides
The Sanger TechniqueThe Sanger Technique Uses Uses
dideoxynucleotidedideoxynucleotides (dideoxyadenine, s (dideoxyadenine, dideoxyguanine, dideoxyguanine, etc)etc)
These are These are molecules that molecules that resemble normal resemble normal nucleotides but nucleotides but lack the normal -lack the normal -OH groupOH group..
Because they lack the -OH (which Because they lack the -OH (which allows nucleotides to join a allows nucleotides to join a growing DNA strand), replication growing DNA strand), replication stops.stops.
Normally, this wouldbe where another phosphateIs attached, but with no -OHgroup, a bond can not form and replication
stops
The Sanger Method RequiresThe Sanger Method Requires Multiple copies of single stranded Multiple copies of single stranded
template DNAtemplate DNA A suitable A suitable primerprimer (a small piece of DNA (a small piece of DNA
that can pair with the template DNA to that can pair with the template DNA to act as a starting point for replication)act as a starting point for replication)
DNA polymeraseDNA polymerase (an enzyme that copies (an enzyme that copies DNA, adding new nucleotides to the 3’ DNA, adding new nucleotides to the 3’ end of the templateend of the template
A ‘pool’ of A ‘pool’ of normal nucleotidesnormal nucleotides A small proportion of A small proportion of dideoxynucleotidesdideoxynucleotides
labeled in some way ( radioactively or labeled in some way ( radioactively or with fluorescent dyes) with fluorescent dyes)
The template DNA pieces are replicated, The template DNA pieces are replicated, incorporating normal nucleotides, but incorporating normal nucleotides, but occasionally and at randomoccasionally and at random dideoxy dideoxy (DD) nucleotides are taken up.(DD) nucleotides are taken up.
This stops replication on that piece of This stops replication on that piece of DNADNA
The result is a The result is a mix of DNA lengthsmix of DNA lengths, each , each ending with a particular labeled ending with a particular labeled DDnucleotide.DDnucleotide.
Because the different lengths ‘travel’ at Because the different lengths ‘travel’ at different rates during electrophoresis, different rates during electrophoresis, their order can be determined.their order can be determined.
Termination during Replication
DNA SEQUENCE 3’
G C A T T G G G A A C C
PRIMER 5’
C G T A
NO OF BASES
1 2 3 4 5 6 7 8 9 10 11 12
G terminated C G T A A C C T T G
C G T A A C C T T G G A terminated
C G T A A Tterminated C G T A A C C T
C G T A A C C T T
C terminated C G T A A C C G T A A C C C G T A A C C C