1. DNA as store of informationmis.agri.cmu.ac.th/download/course/lec_356725_ans725-2...(Mitosis / Miosis) AgBiotech-CMU 2003 Karyotype Cattle Karyotype AgBiotech-CMU 2003 Karyotype

1

Molecular Genetics in Farm AnimalANS 365725

(พันธุศาตรโมเลกุลในปศุสัตว)

1. DNA as store of information

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Organism

Cell

Nucleus

Chromosome

(Karyotype)(Cytogenetics)

(Mitosis / Miosis)

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Karyotype

Cattle Karyotype

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Karyotype

Human karyotype

(Griffiths et al. 2002, An Introduction to Genetic Analysis, 7th edition, p.88)

2

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Chromosome

จํานวนโครโมโซมของสัตวชนิดตางๆ Species จํานวนโครโมโซม (2n)Human 46Dog 78Pig 38Goat 60Sheep 54Cattle 60Horse 64

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Chromosome

Metacentric AcrocentricSubmetacentric Telocentric

Centromere

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Pig chromosomes

Ch1 Ch2 Ch3 Ch4 Ch5 Ch6 Ch7 Ch8 Ch9 Ch10

Ch11 Ch12 Ch13 Ch14 Ch15 Ch16 Ch17 Ch18 Ch X Ch Y

Sex Ch.Autosomal Ch.

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Chromosome

p (short arm)

q (long arm)

1q21.1

Chromosome / long arm / region 2 / band 1 /.subband

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Chromosome

Chromosome ประกอบดวย :

Protein (Histone H1, H2A, H2B, H3, H4)

DNA (sugar, phosphate, bases)

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Chromosome

H1 histone

H2A

H2BH2A

H2BH3H3H4H4

Histone Octamer

Nucleosome

DNA

3


Chromosome


Nucleic acid

DNA (deoxyribonucleic acid)RNA (ribonucleic acid)

pentose sugar (deoxyribose, ribose)

nitrogenous bases:purines: adenine (A), guanine (G), pyrimidine: cytosine (C), thymine (T), uracil (U)

phosphate group


2-Deoxyribose

C

CC

C

O

HOH

OH

HH

OHCH2

Ribose

C

CC

C

O

OHOH

OH

HH

OHCH2

Pentose sugar Nitrogenous bases


N

N

NH2N

NAdenine (A) Guanine (G)

N

N

ON H

NH2N

N

O

NH2

Cytosine (C)N

N

O

OCH3 H

Thymine (T)N

Purine

Pyrimidine

N

O

OH

Uracil (U)N

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2-Deoxyribose

C

CC

C

O

HOH

HH

OHCH2

N

N

NH2N

NAdenine (A)

Nucleoside

Nucleoside = sugar + base

Deoxyadenosine


Nucleotide

Nucleotide = sugar + base + phosphate

Deoxyadenylic acid

2-Deoxyribose

C

CC

C

O

HOH

HH

CH2

N

N

NH2N

NAdenine (A)P

O

OOO-

-

4

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Nucleotide


2-Deoxyribose

C

CC

C

O

HOH

HH

CH2

N

N

NH2N

NAdenine (A)P

O

OOO-

-PO

OOO-

-PO

OOO-

-

Adenosine triphosphate (ATP)


Adenine (A)

C

CC

CO

H

HH

CH2

N

N

NH2N

NPO

OOO-

-

C

CC

CO

H

HH

CH2

N

N

NH2N

NPO

OOO

-

OH

5´ end

3´ end

Phosphodiester bond

(-)

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Hydrogen bond

N

N

N HN

N

N

CH3Thymine (T)

N

H O

O

Adenine (A)

H

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Guanine (G)

N

N

ON H

NHN

Cytosine (C)

NN

H N

O

Hydrogen bond

H

H


P

P

P

P

5´ end

3´ end

P

P

P

P

3´ end

5´ end

Hydrogen bond

5

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Double helix

1 helix = 34 Å ~ 10 bp

Each bp = 3.4 Å

1 base pairs (bp)1 kb = 1000 bp1 Mb = 1000 kb


240 250 260 270 280 290

Abs

orpt

i on

UV wavelength (λ−nm)

Nucleic acids (DNA/RNA) Protein

DNA

Absorption spectrum


240 250 260 270 280 290

Abs

orpt

i on

UV wavelength (λ−nm)

Nucleic acids (DNA/RNA)

1 Optical density (OD) = 50 µg/ml

10 µl DNA990 µl H2O

(1:100)

OD260 nm = 0.20 DNA concentration = ?DNA conc. = 50 x 100 x OD260

= 5000 x OD260= 5000 x 0.20= 1000 µg/ml

M1V1 = M2V21000 ng/µl V1 = 50 ng/µl x 100 µl

V1 = 5000 /1000= 5 µl DNA (stock)

95 µl H2O

DNA conc.(50 ng/ µl)

Total 100 µl

Absorption spectrum

17.06.2003

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Quiz

DNA conc.(25 ng/µl)

Total 100 µl

DNA concentration = ?Stock

solution DNA

25 µl DNA475 µl H2O

OD260 nm = 0.44

DNA stock = ? H2O = ?

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Key

DNA conc.(25 ng/µl)

Total 100 µl

Stock solution DNA

25 µl DNA475µl H2O25 µl in 500

(1:20)

OD260 nm = 0.44

DNA stock = ? H2O = ?

DNA concentration = ?

= 50 µg/ml x 20 x 0.44 = 440 µg/ml (ng/µl)

M1V1 = M2V2440 ng/µl V1 = 25 ng/µl x 100 µl

V1 = 25 ng/µl x 100 µl440 ng/µl

= (2500/440) µl= 5.68 µl DNA= 100-5.68 = 94.32 H2O

6


1 M = 1 mol / l= 1 mM/ml= 1 µM/µl

1mM = 1 mM / l= 1 µM/ ml= 1 nM / µl

1 µM = 1 µM/ l= 1 nM / ml= 1 pM / µl

Mole

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OD measurement

Concentration :

1 OD260 Unit of dsDNA = 50 µg/ml

1 OD260 Unit of ssDNA = 33 µg/ml

Purity : DNA : OD260/OD280 ≥ 1.7−1.8

< contaminated with proteins or phenol> 2 contaminated with RNA

No information about the size of DNA

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OD measurement

Concentration :

1 OD260 Unit of ssRNA = 40 µg/ml

Purity : RNA : OD260/OD280 ≥ 1.9−2.1

< contaminated with proteins or phenol

No information about the size of DNA


Denaturation / renaturation

DNA

Heat / OH - Renaturation

Single-stranded denatured state

DNA

Native state Renatured state


FISH

Fluorescence in situ hybridization


SSCP

DNA

Heat / OH -

Single-stranded denatured stateNative state

ICE

Single stranded conformation polymorphism

7

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Replication of DNA

One original double helix

DNA Polymerase enzyme(5´ to 3´ direction) I, II, III

DNA Polymerase enzyme

DNA Ligase enzyme

Cell


5 ´

3´

3´

Replication of DNA

5 ´Old strand

Old strandA G C

A

TG

CG

A

T

5 ´ 3´

New strand T C G A C

TACG

TC

AGCT

3´5 ´

New strandA G C T

G

OriginalDouble

helix


5 ´

3´

3´

Replication of DNA

5 ´ Old strand

Old strand

5 ´3´

New strand

3´ 5 ´Lagging strand

OriginalDouble

helix

Leading strand


5 ´3´

3 ´5 ´

5 ´ 3´

5 ´3´

Old strand

Old strand

DNA polymerase

Okazaki fragments

DNA polymerase

Ligase enzyme

Replication of DNA


Replication of DNA

19.06.2003

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Replication of DNA

Helicase

DNA Polymerase I, II, III

DNA Ligase enzyme

Primerse (RNA polymerase)

RNA primers

Pol I removes RNA Primer (5´ to 3´ / 3´ to 5´ exonuclease activity = ``proofreading``

Pol II repair DNA damage

Pol III polymerization (5´ to 3´ / 3´ to 5´ exonuclease activity = ``proofreading``

Single strand DNA binding protein (SSBPs)

8

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Replication of DNA

Semiconservative replication

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Polymerase chian reaction

DNAPrimerdNTPs

Taq polymerase

BufferH2O

95 oC30 sec

95 oC3 min

55-60 oC30 sec

72 oC60 sec

35 X

72 oC5 min

10 oC99h

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Replication of DNA

24.06.2003

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Transcription

Genetic information ( stored as nucleic aci)

Protrein / Phenotypedecode

DNA

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Transcription

DNA

mRNA

Protein

Transcription

Translation


Transcription

9

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Transcription

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Transcription

Transcription components:

RNA polymerase (5`to 3`direction)

DNA template

dNTP (A, C, G, U)

No primers

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Transcription components:

Transcription


Transcription

DNA

Promoter Coding sequence of gene3´ 5´

…….GGGCGG..………CCAAT…………….TATAAA….TATA box

(-25 to -30 bp)CAAT box

(-70 to -80 bp)GC box

(-110 bp)

GGGCGC CCAAT TATAAA

Transcription start site (+1)

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Transcription

Transcription: 3 stages

Intiation : RNA polymerase bind to promoter region, open DNA, begins RNA synthesis

Elongation : synthesized in 5´ to 3´ direction

Termination : AAUAAA polyadenyl signal (10-30 bp) beforetermination site

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Transcription

10

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RNA DNA-template Nontemple

Transcription

Conding strand

Non-conding strandAgBiotech-CMU 2003

Transcription

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Transcription

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Transcription

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Transcription

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Transcription

11

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DNA

PromoterCoding sequence of gene

5´ 3´

AAUAAA AAAAAAAAA

AAUAAA AAAAAAAAAM7 G-P-P-PPrimary mRNA

mature mRNA

Transcription

Transcription initiation site

RNA slicing

5´ flank region upstream

3´ downstream

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DNA

PromoterCoding sequence of gene

5´ 3´

AAUAAA AAAAAAAAA

AAUAAA AAAAAAAAAM7 G-P-P-PPrimary mRNA

mature mRNA

Transcription

Transcription initiation site

RNA slicing

5´ flank region upstream

3´ downstream

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RNA splicing

DNA junction GU AG

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AAUAAA AAAAAAAAAM7 G-P-P-P

AAUAAA AAAAAAAAA

Exon

1Ex

on2

Exon

3Ex

on4

Exon

5

Exon

1

Exon

2

Exon

3

Exon

4

Exon

5

Intr

on 1

Intr

on 2

Intr

on 3

Intr

on 4

RNA splicing

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AAUAAA AAAAAAAAAM7 G-P-P-P

AAUAAA AAAAAAAAA

Exon

1Ex

on2

Exon

4

Exon

5

Exon

1

Exon

2

Exon

3

Exon

4

Exon

5

Intr

on 1

Intr

on 3

Intr

on 4

Alternative RNA splicing

Intr

on 2

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Transcription

AAUAAA AAAAAAAAAORF

open reading frameStartcodon

Stopcodon

5´UTR 3´UTR (untranslated region)

Six frame translation

12

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Transcription

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Translation

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Translation

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ACGCCCCAUGAAACGGUAACGAU

Genetic code

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Transcription

DNA RNA ProteinTranscription

Reverse

Transcription

Translation

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cDNA

cDNA : (complementary DNA): exon

: no intron

: code for protein

13

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cDNAAAAAAAAAAAA

Poly-A tail

TTTTTTTTTTTT

Add poly-dT primer TTTTTTTTTTTTAAAAAAAAAAA

TTTTTTTTTTTTAAAAAAAAAAA

TTTTTTTTTTTT

TTTTTTTTTTTTAAAAAAAAAAA

Add reverse transcriptase

dNTPs

TTTTTTTTTTTT

TTTTTTTTTTTT

AAAAAAAAAAA

Remove RNA (NaOH / RNase)

Add DNA polymerase I

Add S1 nuclease to cleave hairpin

5´ 3´

5´3´

3´5´

AAAAAAAAAAA

mRNA

mRNA

single stranded cDNA

dNTPs

Double stranded cDNA

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Transcription

Gene

Transcript

Product

Translation

DNA

RNA

Protein

DNA cloningDNA amplification

DNA sequence

cDNA synthesizedDNA cloning

Protein purificationBiochemical analysis

replication

TranscriptionTranscription

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Transcription

Gene

Transcript

Product

Translation

DNA

RNA

Protein

Transcription

Genome

Transcriptome

Proteome

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Translation

DNA

RNA

Protein

Transcription

Transcriptome Sum of all the transcripts (mRNA) of an organism

ProteomeSum of all the proteins of an organism

Sum of all the genes of organismGenome

Documents

1. DNA as store of informationmis.agri.cmu.ac.th/download/course/lec_356725_ans725-2...(Mitosis / Miosis) AgBiotech-CMU 2003 Karyotype Cattle Karyotype AgBiotech-CMU 2003 Karyotype