T-DNA LB RBauxin cytokin opine Oncogenic genes cloning ch 4-5 (2010).pdfCh 5 Introduction of DNA into Living Cells The basic steps in gene Plasmids cloning: 1 Vector C(h2 3 6 7) Selecting

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Gene Cloning & DNA AnalysisCh pt 4 5Chapter 4-5

LB RBauxin cytokin opine

T-DNA

y p

Oncogenic genes

vir genes ori opine catabolism

Guo-qing Song

Part 1 Basic principles

Gene Cloning & DNA Analysis Part 2 Applications in Research

Part 3 Applications in Biotechnology

Manipulation of Purified DNACh 4

The basic steps in gene The basic steps in gene cloning:

1 Vector (Ch 2 3 6 7)

The range f DNA manipulative enzymes

1. Vector (Ch.2, 3, 6, 7)

2. Digestion (Ch. 4)1. Nuclease

2. Ligases3. Ligation (Ch. 4)

4. Transformation (Ch. 5)

g

3. Polymerases. f m ( . )

5. Selection (Ch. 5)4. Modifying enzymes

5. Topoisomerases

P54-86


The range of DNA manipulative enzymes

Exonucleases1. Nuclease

2. Ligases

• One at a time at the end of DNA molecule

Endonucleases

3. Polymerases

4 M dif in n m

• Break internal phosphodiesterbonds within a DNA molecule

4. Modifying enzymes

5. Topoisomerases

P54-86


(a) An exonuclease


1. Nuclease

2. Ligases

3. Polymerases

4 M dif in n m s

(b) An endonuclease


5. Topoisomerases

P54-86Figure 4.1 (P56)

The reactions catalysed by the two different kinds of nuclease


(a) Bal31


1. Nuclease

2. Ligases

The reactions catalysed by different types of exonuclease

(b) Ex l III

3. Polymerases

4 M dif in n m s

(b) Exonuclease III


5. Topoisomerases

P54-86Figure 4.2 (P57)


(a) S1 nuclease( )


(b) Dn I1. Nuclease

2. Ligases

The reactions catalysed by different types of endonuclease

(b) Dnase I

3. Polymerases

4 M dif in n m s4. Modifying enzymes

5. Topoisomerases(c) A restriction endonuclease

P54-86Figure 4.3 (P58)


(a) Discontinuity repair


1. Nuclease

2. Ligases

The two reactions catalysed by DNA ligase

3. Polymerases

4 M dif in n m s

(b) Joining two molecules


5. Topoisomerases

P54-86Figure 4.4 (P59)


(a) The basic reactionThe range of DNA manipulative enzymes

(a) The basic reaction

1. Nuclease

2. LigasesThe reactions catalysed by DNA polymerase

(b) DNA polymerase I

3. Polymerases

4 M dif in n m s

polymerase(c) The Klenow fragment


5. Topoisomerases(d) Reverse transcriptase

P54-86Figure 4.5 (P59)


(a) Alkaline phosphatase


1. Nuclease

2. LigasesThe reactions catalysed by DNA modifying enzymes

(b) Polynucleotide kinase

3. Polymerases

4 M dif in n m s

modifying enzymes

(A) Terminal deoxynucleotidyl transferase


5. Topoisomerases

P54-86Figure 4.6 (P61)



1. Nuclease

2. Ligases Topoisomerases have yet find a real use in genetic

3. Polymerases

4 M dif in n m s

find a real use in genetic engineering


5. Topoisomerases

P54-86


Restriction endonucleases:Enzymes for cutting DNA


(a) Vector molecules

1. Nuclease

2. Ligases

3. Polymerases

4 M dif in n m s

(b) The DNA molecule containing the gene to be cloned


5. Topoisomerases

P54-86Figure 4.7 (P62)

The need for very precise cutting manipulations in a gene cloning experiment


The discovery & function of restriction endonucleases

(a) Restriction of phage DNAThe range of DNA manipulative enzymes

(a) Restriction of phage DNA

1. Nuclease

2. Ligases

3. Polymerases

4 M dif in n m s

(b) Bacterial DNA is not cleaved


5. Topoisomerases

The function of a restriction endonuclease in a bacterial cell

Figure 4.8 (P63) P54-86


The recognition sequnces of restriction endonucleases


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

P54-86P65


Restriction endonucleasesRestriction endonucleases

(a) Blunt ends


(b) Sticky ends1. Nuclease

2. Ligases

(b) Sticky ends

3. Polymerases

4 M dif in n m s

(c) Same sticky ends pproduced by different restriction endonucleases


5. TopoisomerasesThe ends produced by cleavage of DNA with diferent restriction enzyme Figure 4.9 (P66) P54-86


Restriction endonucleases(a) Cleavage sites on λ DNA

Restriction endonucleases


1. Nuclease

2. Ligases (b) Fragment sizes

3. Polymerases


5. Topoisomerases

Restriction of the λ moleculeFigure 4.10 (P67) P54-86


Restriction endonucleases


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Performing a restriction digest in the laboratory

Figure 4.11 (P68) P54-86


ElectrophoresisElectrophoresis

(a) Standard electrophoresis


1. Nuclease

2. Ligases

3. Polymerases

4 M dif in n m s

(b) Gel electrophoresis


5. Topoisomerases

Figure 4.12 (P71) P54-86


Visualizing DNA


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Visualizing DNA bands in agarose

Figure 4.13 (P72) P54-86


Visualizing DNA


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Visualizing radioactively labelled DNA

Figure 4.14 (P73) P54-86


Vi li i DNAVisualizing DNA


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Radioactively labellingFigure 4.15 (P74) P54-86


The size of DNAThe size of DNA


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Estimation of the size of DNA

Figure 4.16 (P75) P54-86


Isolation of DNA fragments Isolation of DNA fragments based on a restriction map


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Using a restriction map for digestion

Figure 4.17 (P76) P54-86



1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Restriction mapP54-86

Figure 4.18 (P77)


Joining of DNA molecules together


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Ligation: the final step in construction of a recombinant DNA Figure 4.19 (P79) P54-86


Joining of DNA molecules together(a) Ligating blunt ends


1. Nuclease

2. Ligases

(b) Ligating sticky ends

3. Polymerases


5. Topoisomerases

The different joining reactions catalysed by DNA ligase Figure 4.20 (P79) P54-86


Putting sticky ends onto a blunt-ended molecular(a) A typical linker


(b) The use of linkers

(a) typ cal l nker

1. Nuclease

2. Ligases

( )

3. Polymerases


5. Topoisomerases

Linkers and their useFigure 4.21 (P80) P54-86


A possible problem of the use of linkers


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Figure 4.22 (P82) P54-86

A possible problem of the use of linkers


Adaptors and the potential problem with the use


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Figure 4.23 (P82) P54-86

Adaptors and the potential problem with the use


The 5’ and 3’ termini of a polynucleotide


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Figure 4.24 (P83) P54-86

The distinction between the 5’ and 3’ termini of a polynucleotide


Th f d tThe use of adaptors


1. Nuclease

2. Ligases

3. Polymerases


5. Topoisomerases

Figure 4.25 (P84) P54-86The use of adaptors

What is gene cloning? Ch 1


1 Vector (Ch 2 3 6 7)1. Vector (Ch.2, 3, 6, 7)

2. Digestion (Ch. 4)

3. Ligation (Ch. 4)

4. Transformation (Ch. 5). f m ( . )

5. Selection (Ch. 5)

Figure 1.1 (P5)

Introduction of DNA into Living CellsCh 5




Transformation

3. Ligation (Ch. 4)



P87-106

Selection (Ch. 5)





3. Ligation (Ch. 4)



P87-106

Selection (Ch. 5)

TransformationFigure 5.1 (P88)



1 Vector (Ch 2 3 6 7)

Bacterial cells1. Vector (Ch.2, 3, 6, 7)

2. Digestion (Ch. 4)• Bacteria• Phages

3. Ligation (Ch. 4)

4. Transformation (Ch. 5)Non-bacterial cells

. f m ( . )

5. Selection (Ch. 5)• Animal• Plant

P87-106


(a) The product of ligation

The basic steps in gene

(a) The product of ligation

The basic steps in gene cloning:


2. Digestion (Ch. 4) (b) All circular molecules will be cloned

3. Ligation (Ch. 4)



Figure 5.2 (P89) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)The binding and uptake of DNA by a competent 1. Vector (Ch.2, 3, 6, 7)


of DNA by a competent bacterial cell

3. Ligation (Ch. 4)



Figure 5.3 (P91) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

Selecting cells that containing pBR322 plasmids by plating 1. Vector (Ch.2, 3, 6, 7)


p y ponto agar medium containing ampicillinand/or tetracycline

3. Ligation (Ch. 4)



Figure 5.4 (P92) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)Phenotypic expression1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.5 (P93) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

Insertional inactivation(LucZ or antibiotic resistance)1. Vector (Ch.2, 3, 6, 7)


resistance)

3. Ligation (Ch. 4)



Figure 5.6 (P94) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

The cloning vector pBR322

1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.7 (P95) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

Screening for pBR322 recombinants by insertionalinactivation of tetrcycline1. Vector (Ch.2, 3, 6, 7)


inactivation of tetrcyclineresistance gene

3. Ligation (Ch. 4)



Figure 5.8 (P96) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

The cloning vector pUC8

1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.9 (P97) P87-106



Plasmids


1 Vector (Ch 2 3 6 7)

The rationale behind insertional inactivation of the lacZ gene carried by pUC81. Vector (Ch.2, 3, 6, 7)


g y p

3. Ligation (Ch. 4)



Figure 5.10 (P98) P87-106



1 Vector (Ch 2 3 6 7) Phage DNA1. Vector (Ch.2, 3, 6, 7)

2. Digestion (Ch. 4) Transfection=transformation

3. Ligation (Ch. 4)


In vito packaging of λ cloning vectors

. f m ( . )


Figure 5.10 (P98) P87-106



Phage DNA




3. Ligation (Ch. 4)



Figure 5.11 (P100) P87-106

In vito packaging



Phage DNA


1 Vector (Ch 2 3 6 7)Bacteriophage plaques1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.12 (P101) P87-106



Phage DNA


1 Vector (Ch 2 3 6 7)

Strategies for selection of recombinant phage1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.13 (P102) P87-106





3. Ligation (Ch. 4)


Nonbacterial cells

. f m ( . )


P87-106



Nonbacterial cells


1 Vector (Ch 2 3 6 7)

Strategies for inducing new DNA into animal and plant 1. Vector (Ch.2, 3, 6, 7)


cells

3. Ligation (Ch. 4)



Figure 5.14 (P104) P87-106



Nonbacterial cells


1 Vector (Ch 2 3 6 7)

Two physical methods for introducing DNA into cells1. Vector (Ch.2, 3, 6, 7)


3. Ligation (Ch. 4)



Figure 5.15 (P105) P87-106

Documents

T-DNA LB RBauxin cytokin opine Oncogenic genes cloning ch 4-5 (2010).pdfCh 5 Introduction of DNA into Living Cells The basic steps in gene Plasmids cloning: 1 Vector C(h2 3 6 7) Selecting