Section G – Gene manipulation The Beadle-Tatum experiment for the isolation of genetic mutants in...

Section Section G – Gene manipulatiG – Gene manipulationon

The Beadle-Tatum experiment for the isolation of genetic mutants in Neurospora(脉胞菌 ) showed that each mutant had a gene defect that resulted in an enzyme deficiency that prevented the cells from catalyzing a particular metabolic reaction.

“One gene-one enzyme” hypothesis

“One gene-one polypetide” hypothesis

An overview of the flow of information through the cell

Segments of DNA are transcribed

Pre-mRNAs

mRNAs

mRNAs are translated

Proteins

G1 DNA cloning: an overviewG1 DNA cloning: an overview DNA cloning, Hosts and vectors, Subcloning, DNA libraries,

Screening libraries, Analysis of a cloneG2 Preparation of plasmid DNA G2 Preparation of plasmid DNA Plasmids as vectors, Plasmid minipreparation, Alkaline lysis,

Phenol extraction, Ethanol precipitation, Cesium chloride gradient

G3 Restriction enzymes and electrophoreG3 Restriction enzymes and electrophoresis sis

Restriction endonucleases, Recognition sequences, Cohesive ends, Restriction digesis, Agarose gel electrophoresis, Isolation of fragment

G4 Ligation, transformation and analysis G4 Ligation, transformation and analysis of recombinantsof recombinants

DNA ligation, Recombinant DNA molecules, Alkaline phosphatase, Transformation, Selection, Transformation efficiency, Screening transformants, Growth and storage of transfoemants, Gel analysis, Fragment orientation

ContentsContents

G1 DNA cloning: an overview — G1 DNA cloning: an overview — DNA DNA cloningcloning

• DNA cloning facilitates the isolation and manipulation of fragments of an organism’s genome by replicating them independently as part of an autonomous vector.

Question: Why do we have to carry out DNA cloning?Why do we have to carry out DNA cloning?

Applications of DNA cloning

1. Sequencing, hence to derive protein sequence;

2. Isolation and analysis of gene promoter etc;

3. Investigation of protein/enzyme/RNA function in various forms;

4. Identification of mutations;

5. Biotechnology;

6. Transgenic plants and animals ；

7. Gene therapy.

G1 DNA cloning: an overview — G1 DNA cloning: an overview —

Hosts and vectorsHosts and vectors

• Hosts: Hosts: Escherichia coli Escherichia coli Saccharomyces cerivisiaeSaccharomyces cerivisiae

e.g. e.g. E. coli DH5αE. coli DH5α: Host for Blue/White screening utilizing the activity of β-galactosidase (α-complementation) in combination use of pUC vectors. As this strain does not carry lac l, basically IPTG is not needed. Therefore, DH5α allows easy selection of recombinant DNA with X-Gal when constructing gene library or subcloning recombinant plasmid.

• Host organism/cell: where the plasmids get multiplied and propagated faithfully

Vectors: Vectors: Plasmid, Bacteriophages,VirusesPlasmid, Bacteriophages,Viruses BACs, YACs, cosmid, Ti plasmid BACs, YACs, cosmid, Ti plasmid

• A wide variety of natural replicons have the properties of cloning vector.

• General features of a vector for longer term expression of cloned genes in the host

(1) Autonomously replicating DNA independent of host’s genome;

(2) Easily to be isolated from the host cell;(3) Contains at least one selective marker, which allows host cells

containing the vector to be selected among those which do not;

(4) Contains a multiple cloning site (MCS).

Types of vectors

(1) Cloning vectors

(2) Expression vectors

(3) Integration vectors

(4) Viral vectors

(1) Cloning vectors

Allowing the exogenous DNA to be inserted, stored, and manipulated at the DNA level.

E. coli cloning vector: plasmids, bacteriophages ( and M13), plasmid-bacteriophage hybrids (cosmids).

Yeast cloning vector: yeast artificial chromosomes (YACs)

Earlier plasmid

Rop:一种调节蛋白

Versatile cloning plasmid

Phagemid(噬菌粒）

(2) Expression vectors

Allowing the exogenous DNA to be

inserted and expressed. Promoter and

terminator for RNA transcription are

required.

• bacterial expression vectors

• yeast expression vectors

• mammalian expression vectors

(3) Integration vectors Allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is a random insertion by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells.

• Bacterial integration vectors (Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) • Yeast integration vectors• Mammalian integration vectors

(4) Viral vectors

（ 1 ） Bacterial phage: Lambda, M13

（ 2 ） Insect: baculoviruses

（ 3 ） Mammalian viruses:

SV40

Pox virus

Adenovirus

Retroviruses

（ 4 ） Plant viruses: TMV

G1 DNA cloning: an overview — G1 DNA cloning: an overview — SubcloningSubcloning

• SubcloningSubcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality.

• Transfer of a fragment of cloned DNA from one vector to another.

1. Enables us to investigate a short region of a large cloned fragment in more detail.

2. To transfer a gene from one plasmid to a vector designed to express it in a particular species.

Genomic fragment (restriction, PCR), cDNA (insert)

Plasmid preparation

(vector)

Restriction digestion (trimming the DNA ends)

Ligation (join the insert and the

vector) Transformation

(introduce the plasmids into host cells)

Analysis of the recombinants

Electrophoresis (check your DNA)

DNA Cloning: a simplified flow chart

G1 DNA cloning: an overview — G1 DNA cloning: an overview — DNA DNA librarieslibraries

cDNA libraries

DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but yield only the coding region, and not surrounding genomic sequence.

Genomic libraries

Prepared from random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA


Screening libraries Screening libraries

• Searching the interested genes in a DNA librarySearching the interested genes in a DNA library

(1) Colony or plaque hybridization Radiolabeled probes of the interested geneProbes:*An oligonucleotide derived from the sequence of a protein product of the gene * A DNA fragment/oligo from a related gene of another species* PCR product

(2) Identify the protein product of an interested gene(1)Protein activity(2)Western blotting （ Western ） using a specific antibody(3)In vivo expression and functional assay

Plating the cells carrying the library. Colony or plaque lift on membrane and then hybridize with the labeled probe


Analysis of a cloneAnalysis of a clone

(1) Restriction mapping: digestion of th

e plasmids with restriction enzymes.

(2) Sequencing the cloned DNA

Enzymes commonly used in DNA cloning(1) Alkaline phosphotase

You may have to fully understand the function and application of all the listed enzymes if you want to manipulate genes

(2) DNA ligase ( 连接 dsDNA,T4)

(3) DNA pol I 、 Klenow fragment 、 Taq

(4) Exunuclease III

(5) Mung bean nuclease and S1 nuclease

(6) Polynucleotide kinase

(7) Restriction enzymes: e.g. EcoRI, HindIII

(8) Reverse transcriptase

(9) RNase A 、 RNase H

(10) T7, T3 and SP6 RNA polymerases

5’

5’

3’

AAAAACCC-3’

TTTTTP-5’3’-CCCCCCC

AAAAA-3’TTTTTP-5’

Terminal transferasedCTP

(11) Terminal transferase

G2 Preparation of plasmid DNA — G2 Preparation of plasmid DNA —

Plasmids as vectorsPlasmids as vectors

• Plasmids: small, extrachromosomal circular DNA molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells.

(1) Contain an origin of replication and replicate independently

(2) Usually carry a few genes, one of which may confer resistance to antibacterial substance.

G2 Preparation of plasmid DNA — G2 Preparation of plasmid DNA — Plasmid minipreparationPlasmid minipreparation

• A plasmid may be obtained on a small scale for analysis by isolation from a few milliliters of culture, a process known as a minipreparation or miniprep.

G2 Preparation of plasmid DNA — G2 Preparation of plasmid DNA — Alkaline lysisAlkaline lysis

• An alkaline solution of SDS lyses E. coli cells and denatures protein and DNA.

• Neutralization precipitates the chromosomal DNA and most of the protein, leaving plasmid DNA and RNA in solution.


Phenol extractionPhenol extraction

• Extraction with phenol or a phenol-chloroform mixture removes any remaining protein from an alkaline lysate.


Ethanol precipitationEthanol precipitation

• Nucleic acid may be precipitated from solution by the addition of sodium acetate and ethanol, followed by centrifugation.

• The method is used to concentrate the sample.

G2 Preparation of plasmid DNA —G2 Preparation of plasmid DNA —

Cesium chloride gradientCesium chloride gradient

• A CsCl gradient can be used as part of a large-scale plasmid preparation to purify supercoiled plasmid DNA away from protein, RNA and linear or nicked DNA.

1.Growth of the cells containing plasmids;2.Collect the cells by centrifugation;3. Alkaline lysis Resuspend the cells in a buffer solution Cell lysis in lysis buffer containing SDS ，disrupts cell membrane and denatures proteins and NaOH (denatures DNA); Neutralization buffer containing KOAc renaturation of plasmid DNA (supercoiled) and precipitation of denatured proteins and chromosomal DNA. Centrifugation ： plasmid in supernatant (lysate)4.Phenol extraction to get rid of the protein contaminants5. Ethanol precipitation to concentrate the nucleic acids remained (0.3M NaAc, 2-3 vol ethanol). 6. Resuspend in TE buffer

G3 Restriction enzymes and electrophoresis —G3 Restriction enzymes and electrophoresis — Restriction endonucleasesRestriction endonucleases

G3 Restriction enzymes and electrophoresis —G3 Restriction enzymes and electrophoresis —

Recognition sequencesRecognition sequences

Recognize 4-8 bp palindromic sequences. Mos

t commonly used enzymes recognize 6 bp wh

ich occurs at a rate of 46=4096 bp. (44=256 b

p; 48=65536 bp)

5’ GAATTC 3’3’ CTTAAG 5’

e.g. EcoRI site:


Cohesive endsCohesive ends

5’ protruding ends 3’ protruding ends

+

5’-CCCGGG-3’3’-GGGCCC-5’

5’-CCC-OH3’-GGG- p

p -GGG-3’OH-CCC-5’

SmaI

blunt ends

Sticky ends

G3 Restriction enzymes and electrophoresis —G3 Restriction enzymes and electrophoresis — Restriction digesis Restriction digesis

• (1) Commercially available;• (2) Require Mg2+ for enzymatic activity,10M;• (3) Different enzymes, different pHs, NaCl, ot

her solution constituents;• (4) A few hundred nanograms for analysis by

electrophoresis, preparative purposes, a few micrograms;

• (5) 37 , 20ul.℃


Agarose gel electrophoresisAgarose gel electrophoresis

• Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-2%)


Isolation of fragmentIsolation of fragment

insert

1. Restriction digestion2. Agarose gel electrophoresis3. Gel excision and purification4. Ligation with vector5. Transformation

G4 Ligation, transformation and analysis of G4 Ligation, transformation and analysis of

recombinants — recombinants — DNA ligationDNA ligation• To insert a target DNA fragment into a vector,

a method for the covalent joining of DNA molecules is essential.

• DNA ligase: Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.

• To activate the phosphate group for attack by the 3’-OH, the E. coli enzyme uses NAD+, T4 uses ATP.

G4 Ligation, transformation and analysis of G4 Ligation, transformation and analysis of recombinants — recombinants —

Recombinant DNA moleculesRecombinant DNA molecules

• A gene is inserted into a plasmid vector• Target DNA, isolated from an agarose gel;• The target is digested with EcoR , can be ligated Ⅰ

with vector DNA cut with the same enzyme;• The vector should have only one site for cleave wi

th the relevant enzyme;• Circularization of the linear vector is a competing

side reaction, to prepare both target and the vector using a pair of restriction enzymes, they have noncompatible cohesive ends.

G4 Ligation, transformation and analysis of recombinG4 Ligation, transformation and analysis of recombinants — ants — Alkaline phosphataseAlkaline phosphatase

• Treatment of the linear vector molecule with alkaline phophatase will remove the 5’-phosphates and render the vector unable to ligate into a circle without an inserted target, so reducing the proportion of recreated vector in the mixture.

Recombinant DNA molecules


recombinants — recombinants — TransformationTransformation

• Removes the phosphate groups from the 5’-ends of the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligation;

• One phosphate is present to ligate one strand, the remaining nicks will be repaired by cellular mechanisms after transformation.


recombinants — recombinants — SelectionSelection


recombinants — recombinants — Transformation Transformation efficiencyefficiency

• Number of colonies formed on a selective plate per microgram (mg) of input DNA.

• Ranges from 103 to more than 108 ， 105 is adequate for a simple cloning.

• Electroporation is more efficient, up to 109.

G4 Ligation, transformation and analysis of recombinG4 Ligation, transformation and analysis of recombinants — ants — Screening transformantsScreening transformants

• In the case of a simple subcloning experiment, transformants are screened most easily by digesting the DNA from minipreparation of the transformants, followed by analysis on an agarose gel.

G4 Ligation, transformation and analysis of recombinaG4 Ligation, transformation and analysis of recombinants — nts — Growth and storage of transfoemantsGrowth and storage of transfoemants

• Single colonies from a transformation plate are grown in liquid medium, maintaining the antibiotic selection for the plasmid, and a portion of the culture is stored for later use as a frozen glycerol stock.


recombinants — recombinants — Gel analysisGel analysis

• Recombinant plasmids can be distinguished from vectors by size on an agarose gel and by excising the inserted fragment with the same restriction enzyme( s) used to insert it.

G4 Ligation, transformation and analysis of G4 Ligation, transformation and analysis of recombinants — recombinants —

Fragment orientationFragment orientation

• The orientation of the insert in the vector may be determined using an agarose gel by digestion of the plasmid with a restriction enzyme known to cut asymmetrically within the insert sequence.

Multiple choice Multiple choice questionsquestions

1. The presence of a plasmid in a bacterial culture is usually determined by .

A blue-white screening. B growth in the presence of an antibiotic. C a restriction enzyme digest. D agarose gel electrophoresis. 2. The enzyme alkaline phosphatase . A the take-up of a plasmid into a bacterium. B the expression of a gene in a bacterium.C the take-up of a bacteriophage into a bacterium.D the isolation of a plasmid from a bacterium.

3. Transformation is . A the take-up of a plasmid into a bacterium. B the expression of a gene in a bacterium. 、 C the take-up of a bacteriophage into a bacterium. D the isolation of a plasmid from a bacterium. 4. T4 DNA ligase . A requires ATP. B joins double-stranded DNA fragments with an adjacent 3'-pho

sphate and 5'-OH. C requires NADH. D joins single-stranded DNA.

5. In agarose gel electrophoresis . A DNA migrates towards the negative electrod

e. B supercoiled plamids migrate slower than their

nicked counterparts. C larger molecules migrate faster than smaller

molecules.D ethidium bromide can be used to visualize th

e DNA.

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