Section G: Gene manipulationYang Xu, College of Life Sciences Section G-I Gene Manipulation G1 DNA...

Section G: Gene manipulation Yang Xu, College of Life Sciences

Section G-I Gene Manipulation

G1 DNA cloning: an overview G2 Preparation of plasmid DNA G3 Restriction enzymes


G1 DNA Cloning: An Overview

• DNA cloning• Sub-cloning• DNA libraries• Screening libraries

• Vectors• Plasmids• Hosts• Analysis of clone


DNA cloning

• Definition: It is the making process to place the target gene (the gene of interest) in a vector (an autonomously replicating piece of DNA), forming recombinant DNA, which then is placed into another host species.

E ES

target gene Host transformant

Ampr

vector

E

Ori


DNA cloning: Basic Process

(1) Preparation of plasmid DNA containing the cloned target gene.

(2) Digestion of the plasmid with restriction endonucleases.

(3) Separation of the fragments by agarose gel electrophoresis.

(4) Purification of the desired target fragment.

(5) Ligation of the fragments, to form a new recombinant molecule.

(6) Transformation of the ligated plasmid into an E. coli strain.

(7) Selection of transformed bacteria (see Topic G4).

(8) Analysis of recombinant plasmids (see Topic G4).

Example: plasmid (vector) and E. coli (host)


• Definition: It is the process to transfer of a fragment of cloned DNA from one vector to another.

Experimental steps:

1. Preparation: of the plasmid

2. Digestion of the plasmid

3. Separation of the fragments

4. Purification of target fragment

5. Ligation of the fragments

6. Transformation

7. Selection of transformed bacteria.

8. Analysis of plasmids.

+-

Analysis

Subcloning

①

③

②

④

⑥

⑤

⑦

⑧


DNA libraries• Definition: DNA libraries are sets

of DNA clones (vectors/hosts), each of which has been derived from the insert of a different fragment into a vector followed by propagation in the host.

• Classification and features: 1. Genomic libraries: • They are derived from random

fragments of DNA from the genomes of species by shotgun approach;

• The approach may be an inefficient of finding a gene, especially in eukaryotic genomes, where much of the DNA is noncoding.


DNA libraries2. cDNA libraries:

(ProteinCellmRNAcDNAVectorHost)

• They are derived from the mRNA by reverse transcription and are then inserted into a vector;

• cDNA libraries are efficient for finding and cloning a gene, but only the coding region, not the surrounding genomic sequences.


Screening libraries• Definition: It is the process to use a DNA probe to

find the clone that contains the gene interested. • DNA probe: It is a radioactively labeled short DNA

sequence which is partially complementary to a region of the target gene sequence, therefor, the target gene or clone can be detected by its hybridization.

• Making of the probes: 1. The probe sequence might be an oligo-nucleotide

derived from the sequence of the protein product of the gene;

2. From a related gene from another species. 3. An increasingly important method for the

generation of probes is PCR


Vectors-I: Features of vectors• The features of vectors:

– Vectors must normally be capable of being replicated and isolated independently of the host's genome;

– Vectors also have a selectable marker, a gene which allows host cells conferring resistance to a toxin.

– There are some vectors, for example phage (see Topic H2), which can incorporate DNA into the host genome for longer term expression of cloned genes.

B

tetAampr

Ori

pBR322


Vectors-II: Types of vectors• The common vectors: 1. Plasmids:

– Circular plasmid of E. coli: used in E. coli (host);– Yeast episomal plasmids: used in yeast;– Agrobacterium tumefaciens Ti plasmid: in plant.

2. Bacteriophages (viruses infecting bacteria; see Topic R2): – Phage : also been used in E. coli, for cloning larger fragments– Phage M13: used to clone ssDNA used in E. coli.

3. Cosmids (plasmid-bacteriophage hybrids): (see Topic H3). 4. Artificial chromosomes: for cloning huge fragments from

humans.– BAC: Bacterial artificial chromosomes (in E. coli);– YAC: Yeast artificial chromosomes (in Yeast).

5. Virus: for other eukaryotic cells in culture– SV40;– Retroviruses. (see Topic H4).


Plasmids• The first cloning vectors to be used, in the mid

1970s, were natural plasmids originally from E. coli.

• Structure and features: Plasmids are small in size, from 2 to around 200 kb

extrachromosomal circular molecules

which exist in multiple copies (up to a few hundreds)

within the host E. coli cells.

They contain an origin of replication (ori), which enables them to be replicated independently.


Plasmids• Resistance gene: They usually carry a

few genes, one of which may confer resistance to antibacterial substances:

1. The most widely known resistance gene is ampr gene, which encodes the enzyme -lactamase, that degrades penicillin antibiotics such as ampicillin.

2. Another is the tetA gene, which encodes a transmembrane pump able to remove the antibiotic tetracycline from the cell.


Hosts• The common hosts:• E. coli: The initial isolation and analysis of DNA

fragments is almost always carried out using the E. coli as the host (for circular plasmid, phage , phage M13, cosmid, BAC);

• Yeast: It is being used to manipulate very large fragments of the human genome (for episomal plasmid, YAC).

• Other cells:– Agrobacterium tumefaciens (for Ti plamid);– Insect cells (for baculovirus);– Eukaryotic cells (for SV40, retroviruses, shuttle

vectors).


Analysis of clone• Once a clone containing a target gene is identified, the structure

of the cloned fragment may be investigated:

1. further using restriction mapping, the analysis of the fragmentation of the DNA with restriction enzymes (see Topic J1) and by agarose gel electrophoresis using marker of known sizes (see Topic G3),

2. or ultimately by the sequencing of the entire fragment (see Topic J2).

3. The sequence can then be analyzed by comparison with other known sequences from data bases, and the complete sequence of the protein product determined (see Topic J2).


G2 Preparation of Plasmid DNA

• Bacterial culture

• Alkaline lyses

• Phenol extraction

• Ethanol precipitation

• Cesium chloride gradient

Harvest cells

By centrifugationCells

1. Resuspend cell pellet; 2. Add lysozym

3. Add detergent/NaOH; 4.Neutrize withPlasmidRNAProtein Protein, chromosomal DNA

and membranes

Denaturedprotein

Phenol-chloroform

ProteinChromosomal/ inear DNA

Plasmid

RNA

(plasmid + RNA)Aqueous

Pure plasmid

Take aqueous

1. Ethanol precipitate

2. RNase

1. Collect supercoiled plasmid band2. Ethanol precipitate

Extraction:Phenol-chloroform

CsCl gradient

Plasmid mini-preparation

KOAc



G3 Restriction Enzymes and Electrophoresis

• Digestion, separation and purification– Restriction endonucleases– Restriction sequences– Cohesive ends– Restriction digests– Agarose gel electrophoresis


Digestion: Restriction endonucleases

• Function: Restriction-modification systems occur in many bacterial species, and constitute a defense mechanism against the foreign DNA into the cell.

• Structure: Restriction-modification system consist of two parts：1. The first part is a restriction endonuclease, which recognizes a

short, symmetrical DNA sequence (Fig. 1), and hydrolyzes the DNA backbone in each strand at a specific site.

2. The second part is a methylase, which adds a methyl group to a C or A base within the same recognition sequences. This modification protects the host DNA against the endonuclease.


5’-GAATTC-3’3’-CTTAAG-5’

Digestion: Restriction sequences

• Definition: It is a short palindromic sequences, at which restriction enzymes cleave DNA symmetrically in both strands.

• Acting Steps: EcoRI as an example.

– Recognizing: The restriction endonucleases EcoR I acts as a dimer, will only recognize a 6 bp palindromic sequence.

– Cutting: The product of the cutting reaction is two restriction fragments, each with a 5'-end with a phosphate group and a 3'-end with a free hydroxyl group.

5’-G-OH P-AATTC-3’3’-CTTAA-P HO-G-5’

5’-GAATTC-3’3’-CTTAAG-5’

Recognizing

Cutting

annealing

EcoR I


Digestion: Cohesive ends

• Definition: Some of the products of restriction enzyme digestion have protruding ends, and these ends are known as cohesive, or 'sticky' ends.

• Features: Those products of restriction enzyme digestion with protruding ends have a further property: – They can bind to any other end with the same overhanging

sequence, by base pairing (annealing) of the single-stranded tails.

– For example, any fragment formed by an EcoR I cut can anneal to any other fragment formed in the same way, and may subsequently be joined covalently by ligation.

– In fact, in some cases, DNA ends formed by enzymes with different recognition sequences may be compatible


Digestion: Restriction digests-I

• Application: Digestion of plasmid or genomic DNA is carried out with restriction enzymes for analytical or cloning preparation purposes.

• Examples: The digestion of a sample plasmid with two different restriction enzymes, Bam HI and EcoR I.

Plasmidwith gene

X

EE

E

EX

E

B

B B

BamHI

EcoRI

E EB


Digestion: Restriction digests-II

• Reaction system:

– All restriction enzymes require Mg2+ (magnesium)

usually at a concentration of up to 10 mM;

– but different enzymes require different : pHs, NaCl

concentrations or other solution constituents.

• Reaction process: The DNA is incubated at the optimum

temperature (37C) with the enzyme and the appropriate

buffer, in a volume of perhaps 20 l. A dye mixture is then

added to solution, and the sample is loaded on to an agarose

gel.


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Section G: Gene manipulationYang Xu, College of Life Sciences Section G-I Gene Manipulation G1 DNA...

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