Download - DNA and its components
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Chapter 3
Structures and
Functions of
Nucleic Acids
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Nucleic acid
A biopolymercomposed of nucleotides
linked in a linearsequential order through
3,5 phosphodiesterbonds
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Classification of nucleic acid
Ribonucleic acid(RNA) is composed ofribonucleotides.
in nuclei and cytoplasm
participate in the gene expression
Deoxyribonucleic acid (DNA)iscomposed of deoxyribonucleotides.
90% in nuclei and the rest inmitochondria
store and carry genetic information;determine the genotype of cells
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Interesting history
1944: proved DNA is genetic materials(Avery et al.)
1953: discovered DNA double helix (Watson andCrick)
1968: decoded the genetic codes (Nirenberg)
1975: discovered reverse transcriptase(Temin andBaltimore)
1981: invented DNA sequencing method (Gilbert andSanger)
1985: invented PCR technique(Mullis) 1987: launched thehuman genome project
1994: HGP in China 2001: accomplished the draft map of human genome
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Section 1
Chemical Components ofNucleic Acids
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nucleic acid nucleotides
phosphate
nucleosides
pentose
bases
1.1 Molecular Constituents
Nucleic acid can be hydrolyzed intonucleotides by nucleases. The hydrolyzed
nucleic acid has equal quantity of base,
pentose and phosphate.
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N
N
NH
N
12
345
67
8
9
N
N
NH
N
NH2
Adenine (A)
N
NH
NH
N
NH2
O
Guanine (G)
Base: Purine
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N
NH
1
3
2
45
6
Base: Pyrimidine
Cytosine (C)
N
NH
NH2
O
Uracil (U)
NH
NH
O
O
Thymine (T)
NH
NH
O
O
CH3
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Pentose
123
45 OHOCH
2OH
OH OH
-D-ribose
OH
O
CH2
OH
OH
-D-2-deoxyribose
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Ribonucleoside
OHO
CH2
OHOH
N
N
NH2
O
11
Purine N-9 or pyrimidine N-1 is connectedto pentose (or deoxypentose) C-1 through aglycosidic bond.
glycosidic bond
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P
O
O
OH
OH OCH2
OHOH
N
N
NH2
O
A nucleoside (or deoxynucleoside) and aphosphoric acid are linked together through
the 5-phosphoester bond.
Ribonucleotide
phosphoester bond
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base nucleoside nucleotide
guanine guanosine guanosine monophosphate
(GMP)
cytosine cytidine cytidine monophosphate(CMP)
adenine adenosine adenosine monophosphate
(AMP)
uracil uridine uridine monophosphate
(UMP)
(NMP)
Nomenclature
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base nucleoside nucleotide
guanine deoxyguanosine deoxyguanosine monophosphate
(dGMP)
cytosine deoxycytidine deoxycytidine monophosphate
(dCMP)
adenine deoxyadenosine deoxyadenosine monophosphate
(dAMP)
thymine deoxythymidine deoxythymidine monophosphate
(dTMP)
(dNMP)
Nomenclature
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Nucleic
acid base ribose
DNA AGCT deoxyribose
RNA AGCU ribose
Composition of DNA and RNA
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Nucleic acid derivatives
Multiple phosphate nucleotidesadenosine monophosphate (AMP)
adenosine diphosphate (ADP)
adenosine triphosphate (ATP)
N
OCH2O
OHOH
N
NN
NH2
P
O
OH
OH
AMP NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OP
O
OH
OH
ADP
N
OCH2O
OHOH
N
N
N
NH2
P
O
OH
OP
O
OH
OP
O
OH
OH
ATP
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Cyclic ribonucleotide: 3,5-cAMP, 3,5-cGMP, used in signal transduction
N
OCH
2O
OHO
N
NN
NH2
PO
OH
cAMP
Nucleic acid derivatives
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Biologically active systems containingribonucleotide: NAD+, NADP+, CoA-SH
Nucleic acid derivatives
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The
-Patom of the triphosphate group of adNTP attacks the C-3 OH group of a nucleotide
or an existing DNA chain, and forms a 3-
phosphoester bond.
Phosphoester bond formation
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Nucleic acid chain extension
A nucleic acid chain, having a phosphate
group at 5 endand a-OH group at 3 end,
can only be extended from the 3 end.
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Phosphodiester bonds
Alternative
phosphodiester
bonds andpentoses
constitute the 5-
3 backbone ofnucleic acids.
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Section 2
Structures and Functions
of Nucleic Acids
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2.1 Primary Structure The primary structure of DNA and RNA is
defined as the nucleotidesequencein the 5
3 direction.
Since the difference among nucleotides is thebases, the primary structure of DNA and RNA
is actually the base sequence.
The nucleotide chain can be as long asthousands and even more, so that the base
sequence variationscreate phenomenal
genetic information.
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P P
A
P
C
P
C
P
T
P
G
OH
C
P
T
P
A
P
A
5' 3'
pApCpTpGpCpTpApApC-OH 3'
5' ACTGCTAAC 3'
5'
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2.2 Secondary structure
The secondary structure is defined as the
relative spatial positionof all the atoms ofnucleotide residues.
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2.2.a Chargaffs rules The base compositionof DNA generally
varies from one species to another.
DNA isolated from different tissues of the
same species have the same basecomposition.
The base composition of DNA in a givenspecies does not change with its age,
nutritional state, and environmentalvariations.
The molarity of A equals to that of T, and themolarity of G is equal to that of C.
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Molarity of bases
A G C T A/T G/C G+C Pu/Py
E. coli 26.0 24.9 25.2 23.9 1.09 0.99 50.1 1.04
Tuberc
ulosis15.1 34.9 35.4 14.6 1.03 0.99 70.3 1.00
Yeast 31.7 18.3 17.4 32.6 0.97 1.05 35.7 1.00
Cow 29.0 21.2 21.2 28.7 1.01 1.00 42.4 1.01
Pig 29.8 20.7 20.7 29.1 1.02 1.00 41.4 1.01
Human 30.4 19.9 19.9 30.1 1.01 1.00 39.8 1.01
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Historic X-ray diffraction picture
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Building a milestone of life
James Watsonand Francis
Crick proposed a
double helix
model of DNA in
1953.
It symbolized the
new era ofmodern biology.
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Two DNA strands coil together around the
same axisto form a right-handed double
helix (also called duplex).
The two strands run in opposite directions,
i.e., antiparallel.
There are 10 base pairsor 3.4nm per turnand the diameter of the helix is 2.0nm.
2.2.b Double helix of DNA
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Antiparallel
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The hydrophilic
backboneis on
the outsideof theduplex, and the
bases lie in the
inner portionof
the duplex.
Backbone and bases
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The two strands of DNA are stabilized by thebase interactions.
The bases on one strand are paired with the
complementary bases on another strand
through H-bonds, namely GC andA=T.
The paired bases are nearly planarand
perpendicular to helical axis.
Two adjacent base pairs have base-stacking
interactions to further enhance the stability of
the duplex.
Base interactions
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Watson-Crick base pair
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Watson-Crick base pair
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Base-stacking interaction
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Major and minor grooves
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Groove binding
Small molecules like drugs bind in the minorgroove, whereas particular protein motifs can
interact with the major grooves.
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2.2.c Polymorphisms of DNA DNA can resume different forms
depending upon their chemical
microenvironment, such as ionic strength
and relative humidity.
B-form DNA is the predominant structure
in the aqueous environmentof the cells.
A-form and Z-form are also native
structures found in biological systems.
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Feature
A-DNA
B-DNA
Z-DNA
Helix rotation Right-handed Right-handed Left-handed
Base pair per turn 11 10 12
Pitch 2.46nm 3.4nm 4.56nm
Helical diameter 2.55nm 2.0nm 1.84nm
Rise per base pair 0.26nm 0.34nm 0.37nm
Glycosyl formation Anti- Anti- Anti- at C,
syn- at G
Rotation between adjacent
base pair
33 36 -60per
dimer
Relative humidity 75% 92%
Structural features of DNAs
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Triplet DNA
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Hoogsteen base pair
The third strand is using Hoogsteen H-
bonds to pair with bases on the first strand.
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G-quartet DNA
The telomere of DNA
is a G-righ sequence,
such as
5 (TTGGGG)n3
4 G residues
constitute a planewhich is stabilized by
Hoogsteen H-bonds.
G
G
T
T
T
T
TT
G
T G
T
T
G
5'
3'
T
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G-quartet of DNA
Four strands are
arranged in
either parallel or
antiparallel
manner.
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2.3 Supercoil Structure
The two termini of a linear DNA could be
joined to form a circular DNA.
The circular DNA is supercoiled, and
supercoil can be either positive or
negative.
Only the supercoiled DNA demonstrate
biological activities.
2.3.a Supercoil structure
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EM image of supercoiled DNA
Circular DNAs in nature, in general, arenegatively supercoiled.
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2.3.b Prokaryotic DNA Most prokaryotic DNAs are supercoiled. Different regions have different degreesof
supercoiled structures.
About 200 nts will have a supercoil on average.
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2.3.c Eukaryotic DNA DNA in eukaryotic cells is highly packed.
DNA appears in a highly ordered formcalled chromosomes during metaphase,whereas shows a relatively loose form ofchromatinin other phases.
The basic unit of chromatin is nucleosome.
Nucleosomes are composed of DNA andhistone proteins.
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Nucleosome
DNA: ~ 200 bps
Histone: basic
proteins withmany Lys and Arg
residues
H2A (x2),
H2B (x2),
H3 (x2),
H4 (x2)
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Beads on a string
146 bp ofnegatively
supercoiled DNA
winds 1 turns
arounda histone
octomer.
H1 histone bindsto the DNA
spacer.
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The total length
of 46 human
chromosomes is
about 1.7 m, andbecomes 200 nm
long after 5 times
condensation.
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2.4 Functions of DNADNA is fundamental to individual life in
terms of
They are the material basis of lifeinheritance, providing the template for
RNA synthesis.
They are the information basisforbiological actions, carrying the genetic
information.
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DNA is able to replicate itself in a high
fidelity to ensure the genetic informationtransfer from one generation to the next.
DNA can be used as a template to
synthesize RNA (transcription), and RNA
is further used as the template to
synthesize proteins (translation).
DNA posses the inherentand the mutant
properties to create the diversity and the
uniformity of the biological world.
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A geneis defined as a DNA segment
that encodes the genetic information
required to produce functional biological
products.
A gene includes coding regionsas well
as non-coding regions.
Genome is a complete set of genes of a
given species.
Gene and genome
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Section 3
Structures and Functions
of RNA
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Classification
mRNA(messenger RNA): template forprotein synthesis
tRNA(transfer RNA): AA carrier
rRNA(ribosomal RNA): a component ofribosome for protein synthesis
hnRNA(heterogeneous nuclear RNA):
precursor of mRNA snRNA(small nuclei RNA): small RNAs for
processing and transporting hnRNA
Cl f k i RNA
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Classes of eukaryotic RNAs
U i f t
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Unique features
RNA is single stranded, in general.
RNA has self-complementary intrachain
base paring.
The double helical regions of RNA are of
theA-form.
RNA is susceptible to hydrolysis.
3 1 M RNA
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3.1 Messenger RNA
mRNAs constitute 5% of total RNAs.
mRNAs vary significantly in life spans.
hnRNA is the precursor of mRNA.
mRNA is the template for proteinsynthesis, that is, to translate each
genetic codon on mRNA into each AA in
proteins. Each genetic codon is a set ofthree continuous nucleotides on mRNA.
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hnRNA contains intronsand exons. Exons are the sequences encoding
proteins, and introns are non-codingportions.
Splicing process of hnRNA removesintrons and makes mRNA becomematured.
The matured mRNA has special structurefeatures, including 5-capand 3-poly Atail.
mRNA maturation
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5-cap
O
N
NN
N
NH2
O
OCH3O
HH
H
CH2
H
OP
O-
O
O
HN
N
N
O
H2N N O
OH
H H
H
CH2
H
OH
O P
O
O-
CH3
P
O-
O5'
2'3'
5'
mRNA chain
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5-cap addition
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5-cap addition
Methylationcan occur at different siteson G or A.
5-cap can be bound with CBP, benefitingtransportingfrom nucleus to cytoplasm.
5-cap can be recognized by translation
initiation factor. It protects the 5-endfrom exonucleases.
P l A t il
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Poly A tail
20-200 adenine nucleotides at 3 end
a un-translated sequence.
Related with mRNA degradationthat
begins with poly A tail shortening.
Associate with poly A tail binding proteins
for protection
P l A t ili
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Poly A tailing
h RNA li i
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hnRNA
mRNA
hnRNA splicing
intron exon
M t d RNA f k t
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AUG AAA.....AAA
5' non-coding region 3' non-coding region
coding region
5'-cap 3'-poly A tail
UAA
Matured mRNA of eukaryote
3 2 T f RNA
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3.2 Transfer RNA
tRNA is about 15% of total RNA.
tRNA is 65-100 nucleotides long.
There are at least 20 types of tRNA in
one cell.
tRNA serves as an amino acid carrierto
transport AA for protein synthesis.
St t f tRNA
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The overall structure is a cloveleaf,reversed L-shapestructure.
There are three loops (DHU loop,
anticodon loop, TC loop),and fourstems.
The 3-D structure is stabilized by
hydrogen bonds of local intrachainbasepairs on these stems.
Structure of tRNA
Re ersed L shape str ct re
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Reversed L-shape structure
T k it f tRNA
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A tRNA molecule has an amino acid
attachment siteand a template-recognition
site, bridging DNA and protein.
The template-recognition siteis a
sequence of three bases called the
anticodon complementary to the mRNA
codon.
Two key sites of tRNA
Codon and anticodon
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Codon and anticodon
The anticodon
on tRNA pairswith the codon
on mRNA.
Amino acid attachment
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Amino acid attachment The OH group at the
3' end of tRNA linkscovalently to an
amino acid.
Only the attachedAA becomes
activatedandcapable of beingtransported.
Rare Bases
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Rare BasestRNA contains a high portion of unusualbases.
3 3 Ribosomal RNA
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rRNA is the most abundantRNA in cells
(>80%).
rRNA assembles with numerousribosomal proteins to form ribosomes.
3.3 Ribosomal RNA
rRNA provides a proper place for protein
synthesis.
Ribosomes
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Ribosomes associate with mRNA to form aplace for protein synthesis.
Ribosomes of eukaryotes and prokaryotes
are similar in shapes and functions.
Ribosomes
Components of ribosomes
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Components of ribosomes
Prokaryote Eukaryote(E.col i) (Liver of mouse)
Smaller subunit 30s 40s
rRNA 16s 1542 nucleotides 18s 1874 nucleotides
proteins 21 40% of total weight 33 50% of total weight
Larger subunit 50s 60s
rRNA 23s 2940 nucleotides 28s 4718 nucleotides
5s 120 nucleotides 5.85s 160nucleotides
5s 120nucleotides
proteins 31 30% of total weight 49 35% of total weight
Ribosome of E co li
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Ribosome of E. co li
70S ribosome
50S large subunit
23S rRNA 5S rRNA
31 proteins
16S rRNA
21 proteins30S small subunit
Secondary structure of 18S rRNA
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Secondary structure of 18S rRNA
The secondary
structure of rRNA
has many loopsand stems, which
can bind ribosomal
proteins to form anassembly for
protein synthesis.
Ribosomal complex
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m7GpppG AAA...AAA
E
P
A
mRNA
N
Ribosomal complex
Polysomes
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5'
3'
mRNA
Polysomes
EM of polysomes
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EM of polysomes
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General properties
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General properties
Acidity Negative backbone
Viscosity
Concentration and aggregation effects
Optical absorption
UV absorption due to aromatic groups
Thermal stability Disassociation of dsDNA (double-stranded
DNA) into two ssDNAs (single-stranded DNA)
4 1 UV Absorption
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4.1 UV Absorption
Application of OD
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Quantify DNAs or RNAsOD260=1.0 equals to
50g/ml dsDNA
40g/ml ssDNA (or RNA20g/ml oligonucleotide
Determine the purity of nucleic acid samples
pure DNA: OD260/OD280 = 1.8
pure RNA: OD260/OD280 = 2.0
Application of OD260
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Melting curve of dsDNA
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Melting curve of dsDNA
DNA melting
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DNA melting
Melting curve: a graphic presentation ofthe absorbance of dsDNA at 260nm
versus the temperature.
Melting temperature(Tm): thetemperature at which the UV adsorption
reaches the half of the maximum value,
also means that about 50% of the dsDNA
is disassociated into the single-stranded
DNA.
Melting curve shift
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Melting curve shift
Tm of dsDNA depends on its average G+Ccontent. The higher the G+C content, thehigher the Tm.
4 2 Thermal stability
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4.2 Thermal stability Dissociation of dsDNAinto two ssDNAs
is referred to as denaturation.
Denaturation can be partially and
completely.
The nature of the denaturation is the
breakage of H-bonds. Denaturation is a common and
important process in nature.
Denaturation of DNA
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Cooperative unwinding
of DNA strands
Extremes in pH or
high temperature
Denaturation of DNA
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Renaturation of DNA
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Renaturation of DNA
Two separated complementaryDNA
strands can rejoin together to form a double
helical form spontaneouslywhen the
temperature or pH returns to the biological
range. This process is called renaturation
or annealing.
4 3 Hybridization
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4.3 Hybridization The ability of DNA to melt and anneal
reversibly is extremely important.
An association between two differentpolynucleotide chains whose basesequences are complementary is referredto as hybridization.
The stability of the hybridized stranddepends on the complementarydegree.
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Two dsDNA molecules
from different speciesare completely
denutured by heating.
When mixed and slowly
cooled, complementary
DNA strands of each
species will associate
and anneal to formnormal duplexes.
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Two ssDNAs, two ssRNAs, as well as one
ssDNA and one ssRNAcan also behybridized.
Ionic strength, degree of complementary,
temperature, as well as base composition,fragment length of nucleic acids will affect
the hybridization.
It is a common phenomenonin biology,and has been used as a convenient
techniques in medicine and biology.
Target DNA detection
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complementary hybridization
probe: . TAGCTGAG
target: . ATCGACTC
probe: . TAGCTGAG
non-target: . ATCAGCTC
mismatched hybridization
Target DNA detection
Applications
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Applications
Gene structure and expression
Microarray or gene chip
mRNA separation
Gene diagnosis and therapy
PCR technique
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Section 5
Nuclease
Definition and classification
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Nucleases are enzymes that are able tohydrolyze phosphoesterbonds and cleave
DNA or RNA into fragments.
Definition and classification
Deoxyribonuclease (DNase)- specially cleave DNA
Ribonuclease (RNase)- specially cleave RNA
Classification
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Exonucleases
They can cleave terminal nucleotides either
from 5-end or from 3-end, such as
enzymes used in the DNA replication.
Endonucleases
They can cleave internally at either 3 or 5
side of a phosphate group, such as therestriction endonucleases used to construct
the recombinant DNA.
Classification
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5
53
3Endonuclease
Endonuclease
Exonuclease
Exonuclease
Applications
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pp
Participate in DNA synthesis and repair,as well as RNA post-translational
modification
Digest nucleic acids of food for betterabsorption
Degrade the invaded nucleic acids
Construct the recombinant DNA