DNA and its components

8/12/2019 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



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Biologically active systems containingribonucleotide: NAD+, NADP+, CoA-SH



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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

DNA and its components

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