大学分子生物学经典双语课件
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DNA 复性过程遵循二级反应动力学 DNA复性过程中单链消失的速度用公式表示：
-dC/dt=kC2
C/C0=1/(1+kC0t)
其中，C是单位时间的单链DNA的浓度 C0为开始反应时变性解链的单链DNA浓度， t为复性时间 K是复性速度常数（L/mol· s），k取决于阳 离子浓度、温度、pH值、DNA片段大小。
2.1.2.2 Conformation polymorphism of the double helix
Alternative doublehelical structures of DNA
Base Obliquity
helix rise per base pair
bp number per turn
Concept of gene Gene cluster and repetitive sequence Chromosome and nucleosome
Genome
Genetic information flow
2.2 Denaturation, renaturation and hybridization
2.1.3
Triplex DNA
1953, Watson & Crick proposed D.S DNA model and found many redundant hydrogen bonding donor and receptors along big grooves. 1957, Felsenfeld proposed T.S DNA concept
Genome
Genetic information flow
2.1 Structure of DNA
2.1.1
primary structure of DNA Definition：the nucletide residue sequence of the polynucleotide chain； Linkage：3’,5’-phosphodiester bond； Backbone：phosphate + pentose； Direction： 5’ →3’ ；
2.1.4
Tetraplex DNA
1958, Poly(G) X-ray photograph Ring structure of hydrogen Tetrable helix DNA
Formation condition: polyG, 4(dG)
DNA Sculpture at Disneyland
Positive supercoils
Relaxed coils Negative supercoils
Topoisomerases
•Topoisomerases: exist in cell to regulate the level
of supercoiling of DNA molecules
polyA/polyU
polydA/polydT
polyd(AG)/polyd(CT)
1983, Mirkin S.M. found plasmid T.S DNA in pH=4.3 solution
Major 1963 groove Hoogsteen
Trible helix
Py:Pu:Py
Triple Helix DNA
biological activity changed (even lost)； viscosity decreased，粘度 solubility decreased,溶解度 Hyperchromicity: the absorbance of ssDNA is greater than that dsDNA.增色 concentration = 50μg/ml： dNTPs A260 = 1.60 S.S DNA A260 = 1.37 D.S DNA A260 = 1.0
2.1.5
DNA supercoiling
Supercoiling:
Supercoiling is the coiling of the DNA axis upon itself。
Parameters used to express topology properties of DNA:
Linking number(L) Twisting number(T) 缠绕数
Chapter 2 Gene and Chromosome
Contents
1
2 3 4 5 6 7
Structure of DNA Denaturation, renaturation and hybridization
Concept of gene Gene cluster and repetitive sequence Chromosome and nucleosome
2.2.1 Properties of DNA 2.2.1.1 Denaturation
Definition: a number of physical and chemical factors
can lead to the destruction of double-stranded
hydrogen-bonded regions of DNA. the doublestranded nucleic acids are converted to single strands.
94℃
2.2.1.2 Renaturation
Definition：annealing。
Denaturation ▲ D.S DNA ▼ Renaturation S.S DNA
Depends on the collision of complementary S.S. DNA
2.2.1.3 renaturation dynamics
Three dimensional structure of DNA
The Watson-Crick B form DNA Deduced by model building
5’ 3’
Sugar-P backbone is perpendicular to the planar base pairs
charged PO4 on the outside
--
Hydrophobic bases inside
Pitch length
10.5 bp /turn
11Å 20Å
Fig 8-15
5’ 3’
♬ key notes of DNA double helix
Two polynucleotide chains in a DNA double helix; Along the same axis，two chains are wound around each other, resulting in a right-handed double helix; Forms a major groove and a minor groove
2.1.2.1 Stable factors of the double helix
• Base-stacking interaction（hydrophobic effect, the major factor）； • Hydrogen bond between complementary base pairs； • electrovalent bond（between the negative charges carried on the phosphate groups and the positive charges carried on the proteins or metal ions）
transferring the other dsDNA through the break.
Type I topoisomerase
Type II topoisomerase
Contents
1
2 3 4 5 6 7
Structure of DNA Denaturation, renaturation and hybridization
C0t曲线
2.2.2 hybridization
• Definition：the renaturation of regions of complementarity between different nucleic acid strands(DNA or RNA) • Characteristic：sensitive、 specific
•Type I topoisomerase: break one strand of the DNA , and change the linking number in steps of ±1 by passing the other strand through the break. •Type II topoisomerase: break both strands of the DNA , and change the linking number in steps of ±2 by
2.1.2
secondary structure of DNA
----DNA double helix
♬ Experimental basis
X~ray photograph of DNA with high quality： DNA specimens from different species have the same results（constant width; 3.4nm）； Chargaff rules：the rule of the composition of DNA Physical chemistry studies and acid and alkali titrate studies on DNA base ；
Writhing number(W) 扭曲数
L=T+W
Positive supercoils
Negative supercoils
DNA isolated from cell negatively supercoiled by ~5 turns per 100 turns of the helix. Lk / Lk = -0.05
Denaturation factor
pH（>11.3或<5.0） Chemical denaturation (urea、methanal 甲醛) Thermal denaturation Low ion strength低离子强度
Characters of denatured DNA
Helical sense
R handed
diameter （nm） 2.0-2.37 2.55
B-form A-form Z-form
0-1 19-20
0.34 nm 0.23 nm
10 11
R handed
L handed
9
0.38 nm
12
1.8-1.84
பைடு நூலகம்
B-form：relative humidity is 92% A-form：relative devoid of water (under 75%) Z-form：left handed helix H-form：triple helix
The bases lie on the inside，the sugarphosphate backbone is on the outside; The bases are flat structure, lying in pairs perpendicular to the axis
The diameter of the double helix is 2nm; There is a complete turn every 3.4nm, with 10bp per turn.
melting curve and Tm
• Increased temperature can bring about DNA denaturation; • Tm （melting temperature): Temperature when 50% DNA denaturation • Tm is a characteristic constant of DNA
-dC/dt=kC2
C/C0=1/(1+kC0t)
其中，C是单位时间的单链DNA的浓度 C0为开始反应时变性解链的单链DNA浓度， t为复性时间 K是复性速度常数（L/mol· s），k取决于阳 离子浓度、温度、pH值、DNA片段大小。
2.1.2.2 Conformation polymorphism of the double helix
Alternative doublehelical structures of DNA
Base Obliquity
helix rise per base pair
bp number per turn
Concept of gene Gene cluster and repetitive sequence Chromosome and nucleosome
Genome
Genetic information flow
2.2 Denaturation, renaturation and hybridization
2.1.3
Triplex DNA
1953, Watson & Crick proposed D.S DNA model and found many redundant hydrogen bonding donor and receptors along big grooves. 1957, Felsenfeld proposed T.S DNA concept
Genome
Genetic information flow
2.1 Structure of DNA
2.1.1
primary structure of DNA Definition：the nucletide residue sequence of the polynucleotide chain； Linkage：3’,5’-phosphodiester bond； Backbone：phosphate + pentose； Direction： 5’ →3’ ；
2.1.4
Tetraplex DNA
1958, Poly(G) X-ray photograph Ring structure of hydrogen Tetrable helix DNA
Formation condition: polyG, 4(dG)
DNA Sculpture at Disneyland
Positive supercoils
Relaxed coils Negative supercoils
Topoisomerases
•Topoisomerases: exist in cell to regulate the level
of supercoiling of DNA molecules
polyA/polyU
polydA/polydT
polyd(AG)/polyd(CT)
1983, Mirkin S.M. found plasmid T.S DNA in pH=4.3 solution
Major 1963 groove Hoogsteen
Trible helix
Py:Pu:Py
Triple Helix DNA
biological activity changed (even lost)； viscosity decreased，粘度 solubility decreased,溶解度 Hyperchromicity: the absorbance of ssDNA is greater than that dsDNA.增色 concentration = 50μg/ml： dNTPs A260 = 1.60 S.S DNA A260 = 1.37 D.S DNA A260 = 1.0
2.1.5
DNA supercoiling
Supercoiling:
Supercoiling is the coiling of the DNA axis upon itself。
Parameters used to express topology properties of DNA:
Linking number(L) Twisting number(T) 缠绕数
Chapter 2 Gene and Chromosome
Contents
1
2 3 4 5 6 7
Structure of DNA Denaturation, renaturation and hybridization
Concept of gene Gene cluster and repetitive sequence Chromosome and nucleosome
2.2.1 Properties of DNA 2.2.1.1 Denaturation
Definition: a number of physical and chemical factors
can lead to the destruction of double-stranded
hydrogen-bonded regions of DNA. the doublestranded nucleic acids are converted to single strands.
94℃
2.2.1.2 Renaturation
Definition：annealing。
Denaturation ▲ D.S DNA ▼ Renaturation S.S DNA
Depends on the collision of complementary S.S. DNA
2.2.1.3 renaturation dynamics
Three dimensional structure of DNA
The Watson-Crick B form DNA Deduced by model building
5’ 3’
Sugar-P backbone is perpendicular to the planar base pairs
charged PO4 on the outside
--
Hydrophobic bases inside
Pitch length
10.5 bp /turn
11Å 20Å
Fig 8-15
5’ 3’
♬ key notes of DNA double helix
Two polynucleotide chains in a DNA double helix; Along the same axis，two chains are wound around each other, resulting in a right-handed double helix; Forms a major groove and a minor groove
2.1.2.1 Stable factors of the double helix
• Base-stacking interaction（hydrophobic effect, the major factor）； • Hydrogen bond between complementary base pairs； • electrovalent bond（between the negative charges carried on the phosphate groups and the positive charges carried on the proteins or metal ions）
transferring the other dsDNA through the break.
Type I topoisomerase
Type II topoisomerase
Contents
1
2 3 4 5 6 7
Structure of DNA Denaturation, renaturation and hybridization
C0t曲线
2.2.2 hybridization
• Definition：the renaturation of regions of complementarity between different nucleic acid strands(DNA or RNA) • Characteristic：sensitive、 specific
•Type I topoisomerase: break one strand of the DNA , and change the linking number in steps of ±1 by passing the other strand through the break. •Type II topoisomerase: break both strands of the DNA , and change the linking number in steps of ±2 by
2.1.2
secondary structure of DNA
----DNA double helix
♬ Experimental basis
X~ray photograph of DNA with high quality： DNA specimens from different species have the same results（constant width; 3.4nm）； Chargaff rules：the rule of the composition of DNA Physical chemistry studies and acid and alkali titrate studies on DNA base ；
Writhing number(W) 扭曲数
L=T+W
Positive supercoils
Negative supercoils
DNA isolated from cell negatively supercoiled by ~5 turns per 100 turns of the helix. Lk / Lk = -0.05
Denaturation factor
pH（>11.3或<5.0） Chemical denaturation (urea、methanal 甲醛) Thermal denaturation Low ion strength低离子强度
Characters of denatured DNA
Helical sense
R handed
diameter （nm） 2.0-2.37 2.55
B-form A-form Z-form
0-1 19-20
0.34 nm 0.23 nm
10 11
R handed
L handed
9
0.38 nm
12
1.8-1.84
பைடு நூலகம்
B-form：relative humidity is 92% A-form：relative devoid of water (under 75%) Z-form：left handed helix H-form：triple helix
The bases lie on the inside，the sugarphosphate backbone is on the outside; The bases are flat structure, lying in pairs perpendicular to the axis
The diameter of the double helix is 2nm; There is a complete turn every 3.4nm, with 10bp per turn.
melting curve and Tm
• Increased temperature can bring about DNA denaturation; • Tm （melting temperature): Temperature when 50% DNA denaturation • Tm is a characteristic constant of DNA