Nucleic acid
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第六章 核酸化学 Nucleic acid PowerPoint PPT Presentation


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第六章 核酸化学 Nucleic acid. 第一节 概述. 核酸 (nucleic acid) 以核苷酸为基本组成单位的生物大分子,携带和传递遗传信息。 DNA ( D eoxyribo n ucleic a cid) 脱氧核糖核酸 RNA ( R ibo n ucleic a cid) 核糖核酸. 一、核酸的发现和研究工作进展. 1868 年 Fridrich Miescher 从脓细胞中提取“核素” 1944 年 Avery 等人证实DNA是遗传物质 1953年 Watson 和 Crick 发现 DNA 的双螺旋结构

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第六章 核酸化学 Nucleic acid

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Nucleic acid


(nucleic acid)

DNADeoxyribonucleic acid)

RNARibonucleic acid)


  • 1868 Fridrich Miescher

  • 1944AveryDNA

  • 1953WatsonCrickDNA

  • 1966 Nirenberg

  • 1975 TeminBaltimore

  • 1981 GilbertSangerDNA

  • 1985 MullisPCR

  • 1990 (HGP)

  • 1999

  • 2001

  • Post-genome era/OMICS era


90%

(deoxyribonucleic acid, DNA)

(genotype)

(ribonucleic acid, RNA)

DNARNA



  • CHONP(9~11%)

  • SP9-11%

(nucleotide)


(pentose)


1.(base)




260 nm


2. (nucleoside)

C-NC-N

AR, GR, UR, CR

dAR, dGR, dTR, dCR


3. (ribonucleotide)



1

2//

3

  • NDPNTP)

  • cAMPcGMP

  • NADNADPFADCoAAMP

  • UDPGCDP-


ATP

  • ATP ATP,

  • ATP ATP


cAMPcGMP

  • cAMP(3,5-)cGMP(3,5-)

  • cAMPcGMPpH7.4, cAMPcGMP43.9 KJ/molATP



primary structure

1 3, 5

2

  • 4n

  • 5 3

  • 53

3


DNA

  • Chargaff [A] =[T][G] [C]

  • A-TG-C

  • DNAX-


DNA

1. DNAsecondary structure

(1) (Chargaff)

  • [A]=[T][G]=[C]

    [A]+[G]=[T]+[C]()


(2) DNA(double helix model)

  • DNA(DNA)5335


90


2nm0.34 nm103.4 nm

34


DNA:ATGCATGC

DNA


  • (major groove)(minor groove)DNA


  • Z-DNA

  • A-DNA


(3)DNA

DNA

DNA

Na+K+Mg2+DNA


  • DNA1061010

  • 400(basepairbp)DNA1.4106nm1.4mm20nm

  • 6.2107bp2.1cm

  • DNA5100bp 1.7mm


2.DNA

,DNADNA


10nt/<10>10

(L)

(T)DNA

(W)

L=T+W


3. DNA

(nucleosome) DNA

  • ---H2B ,H2A ,H3 ,H4

  • DNA---1.8146 bp)

  • H1DNA54bp


DNA---

5 fold

6 fold

40 fold

7 fold


4. DNA

DNA

DNA


RNA


1. RNA

RNARNA

RNA

RNADNAG C U G-U RNA,

tRNA.


2. RNA

(1) mRNA

  • mRNA57-C2m7GpppNm-

  • 2. mRNA3(polyA)20-250nt)


  • A

  • mRNA

  • mRNA


(2)mRNA

hnRNA (heterogeneous nuclear RNA)

Eukaryote

Polycistronic mRNA

Prokaryote


DNA

DNA

mRNA

hnRNA

mRNA

3mRNA

DNA


3. tRNA

(1) tRNA

10~20% DHU

3 - CCA-OH

5G

TC

(I)

()

(DHU)


3`CCA-OHtRNA

(2) tRNA

-----

  • DHU

  • TC

IV

I

III

-tRNA

II

mRNAanticodon


(2) tRNA

-----L


(3) tRNA


4. rRNA

(1)rRNA:


5. RNA

RNARNAmRNARNA(small non-messenger RNAs, snmRNAs),RNA(non-coding RNA, ncRNA)

  • RNARNARNARNA RNA

  • hnRNArRNAncRNARNAregulatory RNA


  • RNA siRNARNAmRNARNARNA interferenceRNAiRNA


RNAi

1RNA19-21ntsiRNA

2siRNARNARISCRISCmRNAsiRNA3'12mRNA



1DNARNADNA DNARNA

2 DNA0.14mol/LNaCl1~2 mol/LNaClRNA

3

D

D2


4pI 1~2


1

2

RNA2-OHRNA


3

(RNase)(DNase)


Py Pu Py Py Pu Py

RNase I)

-3-

T1RNase T1)

A G U G C A

35-OH


EcoR I

5 GAATTC 3

3 CTTAAG 5

Enzyme number

E. coli

strain

DNase I)

dsDNAssDNA5-

(restriction endonuclease)


  • 260nm


OD260

  • 1. DNARNA

    • OD260=1.0

      • 50g/mlDNA

      • 40g/mlDNARNA

      • 20g/ml

  • 2.

    • DNA: OD260/OD280 = 1.8

    • RNA: OD260/OD280 = 2.0


1DNADNA denaturation

DNADNA


  • DNA,

  • OD260


1DNAOD260


2DNA

DNA80-100

  • melting temperature, TmDNATm


Tm


  • DNA GCTm

pH7.0, 0.165 mol/L NaCl

G+C)% =Tm-69.32.44


2. DNA

  • DNA(renaturation): DNA

  • DNA(annealing)

  • hypochromic effect : DNAOD260


(hybridization)

  • DNADNARNA(heteroduplex)




1. DNA

DNADNP

1MNaClDNP

/

DNA


  • 2. RNA

  • RNaseRNA

  • 0.1DEPC

  • RNARNA

  • RNA

  • 1//RNA

  • 2/RNA


3.

1A50g/ml DNA

40g/ml RNADNA

RNA 670-680nm

DNA+ 595-620nm


9.5%

1g10.5g

DNA

DNA


DNARNA

/DNA

DNA

DNARNA


8M CsCl45000 rpm16h

1.80g/ml1.55g/ml

=CsCl=

=0 +4.22r2 - r02 10-10

0DNA

/

r

G-CDNA

=0.1 xG-C + 1.658


DNA


1.DNA

SangerDNADNADNA


DNA

dNTP


2. DNA

MaxamGilbert

DNA4DNA


4

1GDMSN7

2G+AAGN

3T+CTC

4CC


32P *ACTTCGACAG

/NaClC

*AC

*ACTTC

*ACTTCGA C

*ACTTCGACAG

C+T

*AC

*ACT

*ACT T

*ACTTC

*ACTTCGAC

*ACTTCGACAG

G

*ACTTCG

*ACTTCGACAG

G+A

*A

*ACTTCG

*ACTTCGA

*ACTTCGACA

*ACTTCGACAG

CTACGTAG


(PCR)

  • 1995K.Mullis

  • DNA

  • 4dNTPTaq DNAMg2+

  • 3944560sTm 1min721min


Thermal Cycler Steps

Denature

double-stranded DNA

Anneal

primers to single-stranded DNA

Extend primers,

yielding new double-stranded DNA

Cycling:Repeat

steps 1 through 3 (20 - 40 times)


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