目 录目 录 chapter two structures and functions of nucleic acids
TRANSCRIPT
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目 录
Chapter two
Structures and Functions Structures and Functions of Nucleic Acidsof Nucleic Acids
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目 录
Main contents
1. The components of nucleic acids
2. The structures and functions of DNA3. The structures, and functions of RNA4. The physicochemical properties of nucleic acids
5. Nucleases and Ribozymes
6. Genomics and Human Genome Project
Disease cases
Questions
Demands
Introduction
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目 录
Nucleic acid
Polynucleotide or polymers of nucle
oties which can carry, transmit and exp
ress the genetic information
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目 录
• 1868 Fridrich Miescher the isolation of nuclein from pus leukocytes
• 1944 Avery the confirmation of DNA as genetic molecules
• 1953 Watson and Crick the discovery of DNA double helix structure
• 1968 Nirenberg the discovery of genetic codons
• 1975 Temin and Baltimore reverse transcriptase
• 1981 Gilbert and Sanger the method for DNA sequencing (dideoxy chain terminator sequencing)
• 1985 Mullis the creation of PCR technology
• 1990 human genome project (HGP) start-up in America
• 1994 Chinese human genome project start-up
• 2001 HGP were nearly finished
1.The discovery of nucleic acids and the research advance
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目 录
2. The sorts and distribution of nucleic acids
more than 90% in nucleus , and other found outside of nucleus such as mitochondria, chloroplast, plasmid
Distributed mainly in cytoplasm, minority in nucleus
Deoxyribonucleic acid, DNA
Ribonucleic acid, RNA
Carry genetic information, determine the genotype of individual or cell
Transmit the genetic information and gene expression , sometime work as the carrier of genetic information such as RNA virus
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目 录
The Monomeric Units of Nucleic Acids
Section One
Nucleotides
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目 录
Chemical composition of nucleic acids
1. Element component
C 、 H 、 O 、 N 、 P ( 9~10% )
2. Molecular component
—— base : purines , pyrimidines
—— ribose : ribose , deoxyribose
—— phosphate
3. Building units (monomeric units): nucleotides
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目 录
Chemical composition of nucleic acid
nucleic acid ribonucleotide
ribonucleosidephosphate
base
Pentose
Elementary unit of nucleic acid is nucleotideDNA: dAMP, dGMP, dCMP, dTMPRNA: AMP, GMP, CMP, UMP
The composition of nucleotide
base
Pentose
phosphate
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目 录
Purine
N
N
NH
N1
23
4
567
89
N
N
NH
N
NH2
adenine, A
N
NH
NH
N
NH2
O
guanine, G
Bases
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目 录
N
NH1
32
45
6
Pyrimidine
cytosine, C
N
NH
NH2
O
uracil, U
NH
NH
O
O
thymine, T
NH
NH
O
O
CH3
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目 录
Pentose
( RNA )
1´
2´3´4´
5´OH
O
CH2OH
OH OH
ribose( DNA )
OHO
CH2OH
OH
deoxyribose
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目 录 deoxyribonucleoside : dAR, dGR, dTR, dC
R
1. The structures of nucleotides
(1) The formation of ribonucleoside
It can be formed by glycoside bon
d joined base to ribose (ribonucleosid
e) or deoxyribose (deoxyribonucleosid
e) OHO
CH2
OHOH
N
N
NH2
O
1´
1
ribonucleoside : AR, GR, UR, CR
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目 录
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目 录
(2) The naming of nucleoside or nucleotides
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目 录
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目 录
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Biological active substances containing nucleotide : NAD+ 、 NADP+ 、 CoA-SH 、 FAD etc, containing AMP
nucleotides : NMP , NDP , NTP cyclic nucleotide: cAMP , cGMP
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OH
AMPAMP
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OP
O
OH
OH
ADPADP
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OP
O
OH
OP
O
OH
OH
ATPATP
NO
CH2O
OHO
N
NN
NH2
PO
OH
cAMPcAMP
NADP+NAD+
Some important free nucleotide and Some important free nucleotide and their ramifications in organismstheir ramifications in organisms
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目 录
5´end
3´end
(3)The linkage of nucleotides
The nucleotides ar
e linked together by pho
sphodiester bonds to fo
rm polynucleotides, na
mely nucleic acids
C
G
A
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目 录
5′end
3′end
C
G
A
2. Primary Structure of nucleic acids
Definition The linear sequence of (deoxy)nucleotides ( or base sequences ) in DNA (RNA) is termed primary structure of DNA (RNA).Linkage bondphosphodiester linkage
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A G
P5 P
T
P
G
P
C
P
T
P OH 3
writing
5 pApCpTpGpCpT-OH 3
5 A C T G C T 3 目 录
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目 录
Tautomerism between the lactam and lactim
1.2 Properties of nucleosides and nucleotides
Absorbance of ultraviolet light at 260 nm
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目 录
Section Two
Dimensional Structure
and Function of DNA
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目 录
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目 录
• The secondary structure of DNA----double helix structure
– The research background and historic significance of DNA helix structure
– The key points of DNA double helix structure
• The superhelix structure of DNA and the its composition in chromatin
– The DNA superhelix structure
– The spatial structure of DNA in prokaryote
– DNA constitution in the nucleus of eukaryote cells
• DNA functions
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目 录
5′end
3′end
C
G
A
1. Primary Structure of nucleic acids
Definition The linear sequence of deoxynucleotides ( or base sequences ) in DNA is termed primary structure of DNA.Linkage bondphosphodiester linkage
Backbone: ---Pi-dR-Pi-dR---
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目 录
2. Secondary Structure of DNA----Double Helix Model
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2.1 Background
目 录
The analysis of base composition of DNAChargaff rules
The analysis of chemical and physical da
ta on DNA building model
Primarily x-ray diffraction data collected by Rosalind franklin and Maurice Wilkins
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目 录
Chargaff rulesa. A = T, G = C;b. The base composition is different in different organism species. c. The base composition of different organs in same individual is same.
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目 录
2.2 The key points of DNA Double Helix Model
(1) The DNA double helix is oriented to right-handed running.(2) The two DNA strands are organized in an antiparallel arrangement (i. e. The two strands run in opposite directions, one strand is oriented 5’3’ and the other is oriented 3’ 5’ ).
3’
5’
3’5’
minor groove
majorgroove
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目 录
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目 录
(3) The bases of the
two strands form
hydrogen bonds to
each other; A pairs
with T and G pairs with
C. For each round of
the helix, there are 10
pairs of bases
3’
5’
3’5’
minor groove
majorgroove
2.2 The key points of DNA Double Helix Model
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目 录
Base pairing rules
TA GC
A=T G≡C
Hydrogen bond Hydrogen bond
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(4) The stable forces are hydrogen bonds between base pairs and base stack force.
(5) There are major grooves and minor grooves in DNA double helix.
目 录
3’
5’
3’5’
minor groove
majorgroove
2.2 The key points of DNA Double Helix Model
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目 录
The summary on DNA double helix( Watson, Crick, 1953 )
(1) right-handed oriented double helix, antiparallel
(2) Backbone outside, bases inside
(3) base-pairing, A=T, G≡C
(4) A running of the helix containing 10 pairs of bases
(5) Structure stable depends on the vice-bonds
(6) There are minor grooves and major grooves
hydrogen bonds, base stack force
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目 录
2.4 Po
lymo
rph
ism o
f seco
nd
ary structu
re of
DN
A
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目 录
Forms of DNA
Form Pitch(nm) Residues inclination of base per turn pair from horizontal
A 2.8 11 20°
B 3.4 10 0°
Z 4.5 12 7°
RNA-DNA 2.8 11 20° hybridSource: From Davidson, The biochemistry of the Nucleic Acids, 8thed., revised by Adams, et al. Copyright ©Chapman & Hall, London.
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目 录
( 1 ) Superhelix structure of DNA in prok
aryote ------Circular double stranded superh
elical DNA
2.5 The Superhelix Structure of DNA
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目 录
The nucleosome in eukarytic cells
DNA double helix superhelix
nucleosome chromatin chromoso
me
( 2 ) Superhelix structure of DNA in eukar
yote ------Nucleosomes
The nucleosomes in eukarytic cells
consist of DNA and proteins
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目 录
The elementary unit of chromosome is
nucleosome which consists of DNA an
d histone. Histone includes H1, H2A, H
2B, H3, H4. Two molecules of each H2
A, H2B, H3, H4 constitute the core parti
cle of nucleosome.
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目 录
Core particles
Linkage DNAH1, H2A, H2B, H3, H4 histones
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目 录
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目 录
10 nm fiber
Transverse section
length
wis
e section
30 nm fiber
chromosome
300nm solenoid
chromatin
The folding of nucleosomes, the chromatin condensation, the formation of chromosome
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目 录
Genes consist of regulatory region and large protein-coding segments. Genome is a whole sequence of DNA in an organism. Genetic code: The letters A,G,T and C correspond to the nucleotides found in DNA. They are organized into three-letter code words called codons, and the collection of these makes up the genetic codes.
2.6 Functions of DNA
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目 录
DNA functions:
1. Template of replication
2. Template of transcription
3. To accept some mutations
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目 录
Structures and Functions of RNA
Section Three
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目 录
Structure and Function of RNA
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目 录
The primary structures of RNA
belong to single stranded linear
polynucleotide, but contain part
self-complementary pairing, namely
hairpin structures
1. Structure of RNA
Elementary unit of nucleic acid is nucleotideRNA: AMP, GMP, CMP, UMP
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目 录
Structure Characters of mRNA:(1) 5’-cap sequence: m’GpppNmThe cap functions are to boost the binding of ribosome with mRNA, and to increase the stability of mRNA.
2. Types and Functions of RNAs
2.1 Messenger RNA
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目 录
Cap structure of mRNA 5’-end
Guanine
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目 录
Structure Characters of mRNA:(2) 3’-end sequence: poly AThe 3’-end functions are related to increase the stability of mRNA and the lifetime worked as template for translation.
5’ cap structure 3’ poly A
5’ uncoding region 3’ uncoding regioncoding region
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hnRNA
(intron)
mRNA
(3) Mature process of mRNA *
(exon)
目 录
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目 录
(4) mRNA functions:
As a template of protein
synthesis,
it contains triplet codes.
So, mRNA carries the information
for the primary structure of
proteins, serves as template of
protein synthesis during
translation.
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目 录
DNA
mRNA
蛋白
转录
翻译
Prokaryotic
cells
Transcript
translation
protein
细胞质
细胞核
DNA
内含子外显子
转录
转录后剪接转运
mRNA
hnRNA
翻译蛋白
真核细胞
Transcript
Prost-transcript modification
translationprotein
Eukaryotic cells
extron
intron
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目 录
(1) Composition of transfer RNA, tRNAIt’s the smallest RNA among RNAs, only consists of 70~90 nucleotides.
2.2 Transfer RNA (tRNA)
tRNAs contain some unusual (modified) bases, such as 7-methylguanine, pseudouridine, dehydrouridine,
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目 录
N
NH
NH
N
N
O
CH3
CH3
N
N
NH
N
NHCH2 CH C
CH3
CH3
NH
NH
O
O
H
H
H
H
NH
NH
S
O
N,Ndimethylguanine Dihydrouridine
unusual bases
N6-isopreneadenine4-thiouracil
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目 录
The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids.
(2) Functions of transfer RNA, tRNA
Two roles:
Activating amino acids
Recognizing codons in mRNA
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目 录
◆Contain rare bases, such as DHU, pseudouridine ( ), mG, mC ◆ Stem-loop structure ( local double strands) Secondary structure: cloverleaf pattern ◆ Anticodon in the anticodon loop◆ The base sequence of an anticodon can reversely complement with codon on mRNA.
(3) The structure characters of tRNA
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目 录
The cloverleaf pattern of tRNA ( secondary structure)
Amino acid arm
DHU loop
TΨC loop
Anticodon loop
variable loop
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目 录
5’
3’
AUG
Tyr
The anticodon at the end of a base-paired stem recognizes the triplet nucleotide or codon of the template mRNA.
5’ 3’UAC mRNAGCA CCA UCGAUGMet
CGU
Ala
5’
GGU 5’
Pro
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目 录
The cloverleaf pattern of tRNA ( secondary structure)
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目 录
The tertiary structure of tRNA
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目 录
(1) rRNA structure
(2) rRNA functionsa. A component of ribosomesb. Ribosomes work as the apparatus of protein synthesis
2.3 Ribosomal RNA (rRNA)
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目 录
Eukaryotes
5S rRNA
28S rRNA
5.8S rRNA
18S rRNA
Prokaryotes
5S rRNA
23S rRNA
16S rRNA
(3) rRNA sorts ( dependent on the sedimentation coefficient, S)
Large subunit
small subunit
Large subunit
small subunit
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目 录
The components of ribosomeProkaryote ( E coli.) Eukaryote ( mouse live
r )Small
subunit30S 40S
rRNA 16S 1542 nucleotides 18S 1874 nucleotides
proteins 21 Occupy 40% of total weight
33 Occupy 50% of total weight
Large subunit
50S 60S
rRNA 23S5S
2940 nucleotides120 nucleotides
28S5.85S
5S
4718nucleotides160nucleotides120nucleotides
proteins 31 Occupy 30% of total weight
49 Occupy 35% of total weight
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A large number of discrete, highly conserved, and small stable RNA species are found in eukaryotic cells. The majority of these molecules exist as ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both.
2.4 Other Small Stable RNA
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目 录
The major sorts of RNA and their functions
In the nucleus or in the cytoplasm mitochondrion functions
Ribosomal RNA rRNA mt rRNA the components of ribosomes
Messenger RNA mRNA mt mRNA the template for translationTransfer RNA tRNA mt tRNA activating AA and recognizing codons on mRNAheterogeneous nuclear RNA hnRNA the precursor of mRNA
Small nuclear RNA snRNA related to the splicing and
transfering of hnRNASmall nucleolus RNA SnoRNA related to the processing and modifying of rRNA
Small cytoplast RNA scRNA/7SL-RNA the components of signal
discriminator for proteins
located in endoplasmic reticulu
m
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目 录
Small nuclear RNAs are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, U1, U2, U44, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA.hnRNA ( heterogeneous nuclear RNA )snRNA ( small nuclear RNA)
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The Properties of Nucleic Acid
Section Four
目 录
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目 录
1. The general properties of nucleic acids
1.1 Acidic molecules 1.2 Macromolecules 1.3 High viscosity (DNA)1.4 Ultraviolet absorption ( 260 nm)
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目 录
Extin
ctio
n
coeffi
cie
nt
Ultraviolet absorbent spectrum of various bases (pH 7.0)
Cytosine
Adenine
Guanine
Thymine Uracil
Wavelength
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目 录
2. DNA denaturationDefinitionDouble helix of DNA Single strandThe denaturation factorsheating, chemical reagents, ultraviolet light
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The essence of DNA denaturation is the breaking of hydrogen bonds between the double strands of DNA
Natural DNA Denatural DNA
heating
Cooling slowly
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目 录
The changes of DNA properties after denaturation* Hydrogen linkage broken * A260 ( hyperchromic effect )* Double helix single strand* Tm ( melting temperature )* Tm and G + C Tm = 69.3 + 0.41 ( % G+ C ) < 20 bp, Tm = 4 ( G + C ) + 2 ( A + T )
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(1) The determination of the amount of DNA or RNAOD260=1.0 is corresponded to
50μg/ml double stranded DNA40μg/ml single stranded DNA ( or RNA )20μg/ml oligonucleotides
(2) To estimate the purification of nucleic acid samplesPurified DNA : OD260/OD280 = 1.8
Purified RNA: OD260/OD280 = 2.0
The application of OD260
目 录
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For example: the change of ultraviolet absorption spectrum of DNA induced by denaturation
The ultraviolet absorption spectrum of DNA Hyperchromic effect : the phenomenon of OD260 increase com
panied by DNA denaturation in solution 目 录
Natural state
Denatured state
Ab
sorb
en
t valu
e
Wave length
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Heated denaturation of DNAHeated denaturation of DNA
Melting curve: a graph in which absorbance versus temerature is plotted
目 录
Temperature-optical density profile for DNA
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Tm: Tm is the
temperature at which
50% of DNA in
solution are denatured
by heated , or called
melting temperature,
Tm. The value of Tm
is related to the
contents of G and C.
目 录
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目 录
3. The renaturation of DNA and nucleic acid hybridization
(1) Renaturation Single strand of DNA double helix ( annealing )The best annealing temperature: 25℃ < Tm
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目 录
Hybridization double stranded molecules of DNA-DNA
denaturation renaturation
Various source DNA
Renaturation process
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目 录
(2) Nucleic Acid Hybridization
Definition
The process of forming a double stranded structure from two polynucleotide strands ( either DNA or RNA) from different source is termed hybridization
Hybridization Principle ( To see the above slice or next slice)
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目 录
Double stranded DNA 1
Double stranded DNA 2
Double stranded DNA
heating
heating
single stranded DNA
single stranded DNAHeterogeneous double stranded DNA
cooling
slowly
Cooling slowly
Adding marked single stranded DNA probes
Forming the marked heterogeneous stranded DNA
The denaturation and renaturation of DNA, nucleic acid hybridization
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目 录
(3) The application of Hybridization technology
★ Determining whether a certain sequence
occurs more than once in the DNA of a particular
organism ★ Demonstrating a genetic or evolutionary
relationship between different organisms
★ Determining the number of genes transcribed
in a particular mRNA
★ Determining the location of any given DNA
sequence by annealing with a complementary
polynucleotide probe
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目 录
Nucleasescatalytic RNA and DNA
Section 5
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目 录
(1) DefinitionNucleases are the enzymes capable of degrading nucleic acids. (2) CatalogueDNase Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases RNase Those which specifically hydrolyze ribonucleic acids are ribonucleaes.Endonuclease Exonuclease
1. Nucleases
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目 录
Classification according to the substrates•deoxyribonuclease, DNase
Specially degrade DNA•ribonuclease, RNase
Specially degrade RNA 。–Classification according to the cut sites Endonuclease
restriction endonuclease (RE) and nonRE Exonuclease 5’-3’ exonuclease or 3’-5’ exonuclease
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目 录
2. Ribozymes
There are five classes of ribozymes
Ribozymes are RNA molecules with catalytic activity
★ Three classes carry out self-processing reactions.
★ Two classes ( RNase P and rRNA )are true catalysts that act on separate substrates (containing proteins)
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目 录
3. Deoxyribozymes
These special DNA sequences with catalytic capability are called deoxyribozymes.
Some single-stranded DNA molecules are capable of adopting intricate tertiary structures and performing efficient catalysis.
At present, deoxyribozymes are only found in laboratories.
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目 录
Genomics and Genomics and the Human Genome the Human Genome
ProjectProject
Section 6Section 6
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目 录
1. Characteristics of Eukaryotic Genome DNA
Genomes in eukaryotes are much larger than in prokaryotes
Only a few percent ( 2-4%) of DNA in a mammalian cell may suffice for all genes.
Eukaryotes genes do not overlap but are spaced, on the average, 40 000 bp apart.
There are split genes that consist of exons and introns in eukaryote genomes.
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目 录
2. The human Genome Project
1990, the project started
An international program
Make a sequence map for the total 3 billion bp of human genome
Resource for the investigation of hereditary diseases as well as normal gene structure and expression
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目 录
1. Hemoglobin diseases ( 血红蛋白病 )
Disease Cases
2. Horsebean disease ( 蚕豆病 )
3. X-Fragile Syndrome (X- 脆性综合征 )
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目 录
♣Types of nucleic acids and their functions
Demands
♣ Basic units for nucleic acid composition ♣ The differences between DNA and RNA ♣ Secondary structure characters of DNA♣ Structure characters of mRNA, tRNA, rRNA
♣ Physicochemical properties of DNA, RNA
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Questions :1. Describe the sorts, functions of nucleic acids in nature. What are the characteristics of their chemical components or structures respectively? 2. What are the double helix of DNA? How to prove the structural characters of DNA double helix?
3. Why is it said that DNA are the carriers of genetic substances ? How to demonstrate it ?
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目 录
4. Which important physical and chemical properties of nucleic acids are there that can be utilized ?
5. Illustrate those important free mononucleotides existing in organisms6. Do you know how to
compare DNA with RNA?
7. What are the differences between nucleases and ribozymes?
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目 录
Friedrich Miescher (1844-1895)
Friedrich Miescher worked at the Physiological Laboratory of the University of Basel and in Tübingen and is most well known for his discovery of the nucleic acids.
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目 录
Friedrich Miescher ( 1844 - 1895 ) From an early age Friedrich was recognized as being highly i
ntelligent, but he was shy and introspective - perhaps in part as
the result of a serious hearing impairment he had suffered from
since boyhood. Despite this handicap he took great interest in m
usic.
Miescher had originally intented to study lymphocytes, he col
lected laudable pus from used bandages in the nearby hospital.
He obtained a precipitate from the cell nuclei solution. Obviousl
y this material must have come from the nucleus, and he theref
ore named it nuclein. Using elementary analysis, one of the few
methods available to characterize an unknown compound. He e
xtracted high-molecular-weight DNA from damaged cells event
ually.
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目 录
Oswald T. Avery (1877-1955). Courtesy of The Rockefeller University Archives.
Reich
ard, P
. J. Biol. C
hem
. 2002;277:13355-13362
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目 录
O.T. Oswald Theodore Avery (1877 ~ 1955)
Oswald Theodore Avery (1877-1955) was a distinguished bacter
iologist and research physician and one of the founders of immunoch
emistry. He is best known, however, as a discoverer that deoxyribon
ucleic acid (DNA) serves as genetic material. The Oswald T. Avery C
ollection is a part of the Joshua Lederberg Papers, which are at the
National Library of Medicine and available digitally. The collection
was assembled by Nobel laureate Dr. Lederberg because of the stron
g connection between Dr. Avery's work and his own. The work of Av
ery and his lab, observes Dr. Lederberg, was "the historical platfor
m of modern DNA research" and "betokened the molecular revoluti
on in genetics and biomedical science generally.
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目 录
Rosalin
d F
rank
lin(1920-1958
)
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目 录
Rosalind Franklin ( 1920-1958 ) Born in July of 1920, Rosalind Franklin graduated from Cambridge
University and in 1951 went to work as a research associate for John Randall
at King's College. A chemist by training, Franklin had made original and
essential contributions to the understanding of the structure of graphite and
other carbon compounds. It was Franklin alone whom Randall had given the
task of elucidating DNA's structure.
The technique with which Rosalind Franklin set out to do this is called X-
ray crystallography. With this technique, the locations of atoms in any crystal
can be precisely mapped by looking at the image of the crystal under an X-
ray beam. By the early 1950s, scientists were just learning how to use this
technique to study biological molecules. Rosalind Franklin applied her
chemist's expertise to the unwieldy DNA molecule. After complicated
analysis, she discovered (and was the first to state) that the sugar-phosphate
backbone of DNA lies on the outside of the molecule. She also elucidated the
basic helical structure of the molecule.
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目 录
More advanced reading:More advanced reading:
• Nelson, D. L., and Cox, M. M. (2000) Lehninger Principles of Biochemistry, third edition, Worth Publishers.
• Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002) Biochemistry, Fifth edition, W. H. Freeman and Company, New York.
• Mathews, C. K., van Holde, K. E., and Ahern, K. G. (2000) Biochemistry, third edition, Benjamin/Cummings (www.aw-bc.com/mathews/).