中國(guó)農(nóng)業(yè)大學(xué)分子生物學(xué)課件

1、Chapter 2 DNA Structure and properties,§ 2-1 DNA is the Genetic Material§ 2-2 The Chemical Nature of Polynucleotides§ 2-3 DNA is a Double Helix§ 2-4 Polymorphism of DNA Structure§ 2-5 D

2、enaturation and Renaturation § 2-6 Supercoiled DNA and Topoisomerase § 2-7 Restriction Enzymes and DNA Sequencing,§ 2-1 DNA is the genetic material,,deoxyribonucleic acid (DNA) DNA is the storehouse,

3、or cell library, that contains all the information required to build the cells and tissues of an organism.,,1.The Discovery of DNA Friedrich Miescher(1869) discovered in the cell nucleus a mixture of compounds—nuclein.

4、Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins,2. Transformation in Bacteria,Key experiments done by Frederick Griffith in 1928Observed change in Streptococcus pneumoniae — from virulent (S) smo

5、oth colonies where bacterial had capsules, to avirulent (R) rough colonies without capsulesHeat-killed virulent colonies could transform avirulent colonies to virulent ones,DNA: The Transforming Material,In 1944 Avery u

6、sed a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substanceTechniques used excluded both protein and RNA as the chemical agent of transformationOthe

7、r treatments verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent,Griffith, 1928; Avery et al., 1944,3. DNA Confirmation,In 1952, Hershey and Chase demonstrated that bact

8、eriophage infection comes from DNABy 1953 Watson & Crick published the double-helical model of DNA structure and Chargaff had shown that the 4 bases were not present in equal proportions,1952 Martha Chase & Alf

9、red HersheyUsed phages in which the protein was labeled with 35S and the DNA with 32P for the final proof that DNA is the molecule of heredity.,DNA is a main genetic material,RNA is genetic material also,RNA VirusViroi

10、d,,是否存在核酸之外的 其他遺傳物質(zhì)？,Is the protein genetic material ?,復(fù)制 ?表面遺傳信息 ?,,高度純化的病體腦組織的感染性粒子,,,可被蛋白酶K抑制,不被核酸酶和輻射滅活,不含大于100nt的核酸,28kD 的疏水性糖蛋白 由核基因組基因 PrP（Prion related Protein）編碼,Prion (proteina

11、ccous infections particle) 傳染性病原蛋白顆粒引起的風(fēng)波,,Conclusion:Genes are made of nucleic acid, usually DNASome simple genetic systems such as viruses have RNA genes,DNA作為遺傳物質(zhì)的優(yōu)點(diǎn) (自然選擇的優(yōu)勢(shì)),★ 儲(chǔ)存遺傳信息量大,&

12、#167; 2-2 The Chemical Nature of Polynucleotides,,Levene, Jacobs, et al. characterized basic composition of DNA and RNAA nucleic acid consists of a chemically linked sequence of subunits.,Each subunit contains a nitroge

13、nous base, a pentose sugar, and a phosphate group.,Nucleic Acid,Mono-phosphate,Nucleoside,Deoxy-ribose ( Ribose ),Base,A nucleic acid consists of a polynucleotide chain. nitrogenous base is linked to the sugar. DNA takes

14、 its name from its sugar (2-deoxyribose); RNA is named for its sugar (ribose). The difference is that the sugar in RNA has an OH group at the 2 position of the pentose ring.,1. Nitrogenous base,Nucleotide (Nt) basic unit

15、,Each nucleic acid contains 4 types of base. The same two purines, adenine and guanine, are present in both DNA and RNA. The two pyrimidines in DNA are cytosine and thymine; in RNA uracil is found instead of thymine. Th

16、e only difference between uracil and thymine is the presence of a methyl substituent at position C5. The bases are usually referred to by their initial letters. DNA contains A, G, C, T, while RNA contains A, G, C, U,Two

17、 types of pentose are found in nucleic acids.They distinguish DNA and RNA.,2. Pentose sugar,The difference lies in the absence/presence of the hydroxyl group at position 2 of the sugar ring.,The nitrogenous base is link

18、ed to position 1 on the pentose ring by a glycosidic bond from N1 of pyrimidines or N9 of purines.,NucleosideNucleotide,Nucleosides ( 核苷 ) : base + sugar = nucleosideRNA: ribonucleosides or just nucleosides;DNA: 2’-d

19、eoxyribonucleosides or just deoxynucleosidesNucleotides ( 核苷酸 ) : base + sugar + phosphate = nucleotidesThe nucleoside 5’-triphosphates (NTPs or dNTPs) are respectively the building blocks of polymeric RNA and DNA,

20、The polynucleotide chain is constructed by linking the 5′ position of one pentose ring to the 3′ position of the next pentose ring via a phosphate group. So the sugar-phosphate backbone is said to consist of 5′-3′ phosph

21、odiester linkages. The nitrogenous bases "stick out" from the backbone.,Phosphodiester bonds (磷酸二脂鍵) : Covalent linkage of a phosphate group between the 5’-hydroxyl of a ribose and the 3’-hydroxyl of the next.

22、At neutral pH, each phosphate group has a single negative charge, this is why nucleic acids are termed acids; they are the anions of strong acids.Nucleic acids are thus highly charged polymers.,The terminal nucleotide

23、at one end of the chain has a free 5′ group; the terminal nucleotide at the other end has a free 3′ group. It is conventional to write nucleic acid sequences in the 5′→3′ direction-that is, from the 5′ terminus at the le

24、ft to the 3′ terminus at the right.,DNA/RNA sequence: The nucleic acid sequence is the sequence of bases A, C, G, T/U in the DNA/RNA chain. The sequence is conventionally written fr

25、om the free 5’- to the free 3’- end of the molecule.5’- ATTAGCTC- 3’(DNA) 5’- AUAGCUUGA-3’ (RNA),§2-3 DNA is a Double Helix,1. Chargaff’s rulesErwin Chargaff’s studies of the base compositions of DNAs from variou

26、s sources revealed that the content of purines was always roughly equal to the content of pyrimidines.,Erwin Chargaff 1949 DNA是由四種脫氧核苷酸(nucleotide)，就是腺嘌呤(adenine)、鳥(niǎo)嘌呤(guanine)胸腺嘧啶(thymidine)和胞嘧啶(cytocine)組成的，且在不

27、同物種中四種核苷酸的比率不同。但A 與T的量相等，G與C 的量相等，即A=T；G=C，這就是所謂的Chargaff規(guī)則（Chargaff's rules)。,2. The double helix model for DNA by Watson and Crick,,The double helix model 1). X-ray diffraction data showed that DNA has the form of

28、 a regular helix, making a complete turn every 34 Å (3.4 nm), with a diameter of ~20 Å (2 nm). Since the distance between adjacent nucleotides is 3.4 Å, there must be 10 nucleotides per turn. 2). The den

29、sity of DNA suggests that the helix must contain two polynucleotide chains. The constant diameter of the helix can be explained if the bases in each chain face inward and are restricted so that a purine is always opposit

30、e a pyrimidine, avoiding partnerships of purine-purine (too thick) or pyrimidine-pyrimidine (too thin).,3). The proportion of G is always the same as the proportion of C in DNA, and the proportion of A is always the same

31、 as that of T. So the composition of any DNA can be described by the proportion of its bases that is G + C. This ranges from 26% to 74% for different species. Watson and Crick proposed that the two polynucleotide chains

32、 in the double helix associate by hydrogen bonding between the nitrogenous bases. G can hydrogen bond specifically only with C, while A can bond specifically only with T. These reactions are described as base pairing, an

33、d the paired bases (G with C, or A with T) are said to be complementary.,The model requires the two polynucleotide chains to run in opposite directions (antiparallel). Looking along the helix, therefore, one strand runs

34、in the 5′-3′ direction, while its partner runs 3′-5′.,The bases lie on the inside. They are flat structures, lying in pairs perpendicular to the axis of the helix. Consider the double helix in terms of a spiral staircase

35、: the base pairs form the treads. Proceeding along the helix, bases are stacked above one another, in a sense like a pile of plates.,Each base pair is rotated ~36º around the axis of the helix relative to the next b

36、ase pair. So ~10 base pairs make a complete turn of 360º. The twisting of the two strands around one another forms a double helix with a narrow groove (~12 Å across) and a wide groove (~22 Å across). The

37、 double helix is right-handed; the turns run clockwise looking along the helical axis. These features represent the accepted model for what is known as the B-form of DNA.,,flash,,§ 2-4 Polymorphism of DNA Structure,

38、1. The double helix exists in multiple conformation 1). Right-Handed Helix： B，A，C，D，E 2). Left-handed helix： Z-DNA,A-DNA,B-DNA,Z-DNA,Relative humidity

39、 92% 75%Salt sodium sodium,B-DNA A-DNA,,The A form has 11 base pairs per turn. Its major groove is narrower and deeper than that of

40、B form, and its minor groove is broader and shallower.unclear if pure A-form DNA exists within cells. DNA-RNA hybrid double helices all double-helical RNA regions,Take A-form,Structural properties of A、B and Z DNA

41、,2. Z-DNA Alexander Rich et al (MIT)Double-stranded DNA containing strands of alternating purines and pyrimidines. e.g. poly[dG-dC] poly[dC-dG] -GCGCGCGC- -CGCGCGCG- -

42、 m5C GAT m5C G- - G m5CTA G m5C-living cells contain a small proportion of Z-DNAFunction of Z-DNA: regulate gene expression,Z-DNA in Drosophila chromosome proved by anti-

43、Z antibody,Immunologicalslid,Cytological slid,,,,3. Inverted repeatshairpinWhen a sequence of bases is followed by a complementary sequence nearby in the same molecule, the chain may fold back on itself to generate an

44、 antiparallel duplex structure, call a hairpin. It consists of a base-paired, double-helical region, the stem, with a loop of unpaired bases at one end.,hairpin,Inverted repeats:The sequence in a doubled DNA consists of

45、 two copies of an identical sequence present in the reverse orientation. They called inverted repeats.e.g. 5’- GCTTTT……AAAAGC -3’ 3’- CGAAAA……TTTTCG -5’The sequence of the inverted repeats together is

46、 called a palindrome (回文結(jié)構(gòu)). It is defined as a sequence of duplex DNA that is the same when either of its strands is read in a defined direction.,A palindromic sequence is formally described as a region of dyad symmetr

47、y（雙重對(duì)稱區(qū)），in which the axis of symmetry separates the inverted repeats.,GGTCCA,ACCTGG,,The two copies of an inverted repeat need not necessarily be contiguous.,GGTNNCCANN,NNACCNNTGG,,palindrome,§ 2-5 Denaturation

48、 and Renaturation Chemical and Physical Properties of Nucleic Acids Stability of nucleic acids Although it might seem obvious that DNA double strands and RNA structures are stabilized by hydrogen bonding, t

49、his is not the case. H-bonds determine the specificity of the base pairing, but the stability of a nucleic acid helix is the result of hydrophobic and stacking interaction between the stacked base pairs.,,,Effect of aci

50、dIn strong acid and at elevated temperatures, nucleic acids are hydrolyzed completely to their constituents: bases, ribose or deoxyribose and phosphate.In more dilute mineral acid, pH3-4, the most easily hydrolyzed bon

51、ds are selectively broken.Effect of alkaliIncreasing pH above pH8, specific hydrogen bonding broken, dsDNA ssDNABecause of the presence of the 2'-OH group, RNA more susceptible to alkali.In high pH, phospho

52、diester bond (backbone) is broken. RNA fragments,,,Chemical denaturationAt neutral pH, urea and formamide can cause the denaturation of DNA or RNA by disrupting the hydrophobic forces between the stacked bases.Eff

53、ect of mechanical forces DNA is a relatively stiff molecule .long DNA molecular can easily be damaged by shearing forces, or by sonication (high-intensity ultrasound).They merely reduce the length of the double-strand

54、ed molecules in the solution.,,A crucial property of the double helix is the ability to separate the two strands without disrupting covalent bonds. This makes it possible for the strands to separate and reform under phys

55、iological conditions at the (very rapid) rates needed to sustain genetic functions. The specificity of the process is determined by complementary base pairing.,2. Denaturation and Renaturation,DNA的呼吸作用Denaturation: Den

56、aturation of DNA or RNA describes its conversion from the double-stranded to the single-stranded state.The complementary strands of the double helix can be made to come apart when a solution of DNA is heated above physi

57、ological temperature (to near 100?C) or under condition of high pH, a process known as denaturation.,Hyperchromicity( 增色效應(yīng)),Hyperchromicity: When the temperature of a solution of DNA is raised to near the boiling point o

58、f water, the absorbance at 260 nm markedly increases, a phenomenon known as hyperchromicity. This increase is that duplex DNA absorbs less ultraviolet light by about 40% than do individual DNA chains.This hyperchromici

59、ty is due to base stacking, which diminishes the capacity of the bases in duplex DNA to absorb ultraviolet light.,當(dāng)濃度都為50μg/ml時(shí)，Double strained DNA A260=1.00Single strained DNA (完全變性DNA)

60、 A260=1.37單核苷酸的等比例混合物 A260=1.60Tm : Melting temperature of DNA is the mid-point of the transition when duplex DNA to denatured by heating to separate into single strands.,The Tm of DNA i

61、s a characteristic of each DNA. Tm is largely determined by:1. GC content2. Ionic strength3. pH,Tm ↑,pH ~ 12,酮基 → 烯醇基,pH ~ 2-3,NH2 → NH2+ (質(zhì)子化),改變氫鍵的形成與結(jié)合力,☆極端pH條件的影響,,一切減弱氫鍵， 堿基堆積力的因素 均將使Tm 值降

62、低,,Renaturation Renaturation is the reassociation of denatured complementary single strands of a DNA double helix.,Renaturation is determined by:Temperature of solution Concentration of DNAComplexity of DNA,Renaturat

63、ion kinetic (復(fù)性動(dòng)力學(xué))DNA復(fù)性是一種雙分子二級(jí)反應(yīng)，單鏈消失的速度可表示為：,C：?jiǎn)捂淒NA濃度 t：時(shí)間 k：復(fù)性速度常數(shù) Co: DNA濃度,,當(dāng)t=0時(shí)，C=C0,,在上式中，當(dāng)t=0時(shí)，C=C0，表明所有的DNA都是單鏈。,復(fù)性的分?jǐn)?shù)C/ C0是起始濃度和經(jīng)過(guò)時(shí)間的乘積Cot的函數(shù)，這個(gè)函數(shù)繪成的曲線，稱為Cot曲線。,,當(dāng)反應(yīng)完成

64、一半時(shí)：,,,反應(yīng)進(jìn)行至一半時(shí)的Cot，即C0t1/2，其意義為半數(shù)變性DNA再結(jié)合時(shí)的Cot值；反映復(fù)性進(jìn)行至一半時(shí)所需的時(shí)間與DNA濃度間的關(guān)系。任何一個(gè)DNA的復(fù)性都可用C0t1/2描述（核苷酸mole.sec.L-1），值越大表明反應(yīng)越慢，DNA的復(fù)性遵循C0t曲線。,The concept of base pairing is central to all processes involving nucleic acids.

65、Disruption of the base pairs is a crucial aspect of the function of a double-stranded molecule, while the ability to form base pairs is essential for the activity of a single-stranded nucleic acid.,3. Nucleic acids hybri

66、dize by base pairing,The principle of the hybridization reaction is to expose two single-stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms.,Application

67、 of hybridization Library ScreeningSouthern BlottingNorthern Blotting,,,,λphage 為載體構(gòu)建Genomic DNA library,λDNA,,Genomic DNA,,,,,,§ 2-6 Supercoiled DNA and Topoisomerase,Supercoiling of the DNA double helix results

68、 when it is coiled about itself in space (like twisting a rubber band). The supercoiling creates a tension in the double helix that changes its structure.,,Spiral writhe,Interwound writhe,Positive supercoiling, when the

69、DNA is twisted in space in the same sense as the strands are wound around one another, causes the double helix to be more tightly wound. –Left-handedNegative supercoiling, when the DNA is twisted in space in the opposit

70、e sense from the internal winding of the strands, causes the double helix to be less tightly wound. Negative supercoiling can be thought of as creating tension in the DNA that is relieved by unwinding the double helix. –