Download DNA cloning

Section G – Gene manipulation
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
Contents
G1 DNA cloning: an overview
DNA cloning, Hosts and vectors, Subcloning, DNA libraries,
Screening libraries, Analysis of a clone
G2 Preparation of plasmid DNA
Plasmids as vectors, Plasmid minipreparation, Alkaline lysis,
Phenol extraction, Ethanol precipitation, Cesium chloride
gradient
G3 Restriction enzymes and
electrophoresis
Restriction endonucleases, Recognition sequences, Cohesive
ends, Restriction digesis, Agarose gel electrophoresis,
Isolation of fragment
G4 Ligation, transformation and analysis
of recombinants
DNA ligation, Recombinant DNA molecules, Alkaline
phosphatase, Transformation, Selection, Transformation
efficiency, Screening transformants, Growth and storage of
transfoemants, Gel analysis, Fragment orientation
G1 DNA cloning: an overview —
DNA cloning
• 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?
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 —
Hosts and vectors
• Hosts: Escherichia coli
Saccharomyces cerivisiae
e.g. E. 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: Plasmid, Bacteriophages,Viruses
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
(l and M13), plasmid-bacteriophage l 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 —
Subcloning
• Subcloning 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.
DNA Cloning: a simplified flow chart
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)
G1 DNA cloning: an overview —
DNA libraries
Genomic libraries
cDNA libraries
Prepared from random
fragments of genomic
DNA, which may be
inefficient to find a gene
because of the huge
abundance of the noncoding DNA
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.
G1 DNA cloning: an overview —
Screening libraries
• Searching the interested genes in a DNA library
(1) Colony or plaque hybridization
Radiolabeled probes of the interested gene
Probes:
*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
G1 DNA cloning: an overview —
Analysis of a clone
(1) Restriction mapping: digestion of the
plasmids with restriction enzymes.
(2) Sequencing the cloned DNA
You may have to fully understand the
function and application of all the listed
enzymes if you want to manipulate genes
Enzymes commonly used in DNA cloning
(1) Alkaline phosphotase
(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’
3’
5’
3’-CCCCCCC
AAAAA-3’
TTTTTP-5’
Terminal transferase
dCTP
(11) Terminal transferase
AAAAACCC-3’
TTTTTP-5’
G2 Preparation of plasmid DNA —
Plasmids 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 —
Plasmid 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 —
Alkaline 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.
G2 Preparation of plasmid DNA —
Phenol extraction
• Extraction with phenol or a phenolchloroform mixture removes any remaining
protein from an alkaline lysate.
G2 Preparation of plasmid DNA —
Ethanol 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 —
Cesium 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 contaminants
5. 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 —
Restriction endonucleases
G3 Restriction enzymes and electrophoresis —
Recognition sequences
Recognize 4-8 bp palindromic sequences.
Most commonly used enzymes recognize 6
bp which occurs at a rate of 46=4096 bp.
(44=256 bp; 48=65536 bp)
e.g. EcoRI site:
5’ GAATTC 3’
3’ CTTAAG 5’
G3 Restriction enzymes and electrophoresis —
Cohesive ends
5’ protruding ends
3’ protruding ends
Sticky ends
5’-CCCGGG-3’
3’-GGGCCC-5’
SmaI
5’-CCC-OH
3’-GGG- p
blunt ends
+
p -GGG-3’
OH-CCC-5’
G3 Restriction enzymes and electrophoresis —
Restriction digesis
• (1) Commercially available;
• (2) Require Mg2+ for enzymatic activity,10M;
• (3) Different enzymes, different pHs, NaCl,
other solution constituents;
• (4) A few hundred nanograms for analysis by
electrophoresis, preparative purposes, a few
micrograms;
• (5) 37℃ , 20ul.
G3 Restriction enzymes and electrophoresis —
Agarose gel electrophoresis
• Agarose: a polysaccharide derived from
seaweed, which forms a solid gel when
dissolved in aqueous solution (0.5%-2%)
G3 Restriction enzymes and electrophoresis —
Isolation of fragment
1.
2.
3.
4.
5.
Restriction digestion
Agarose gel electrophoresis
Gel excision and purification
Ligation with vector
Transformation
insert
G4 Ligation, transformation and analysis of
recombinants —
DNA 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
recombinants —
Recombinant DNA molecules
• A gene is inserted into a plasmid vector
•
Target DNA, isolated from an agarose gel;
•
The target is digested with EcoRⅠ , can be ligated
with vector DNA cut with the same enzyme;
•
The vector should have only one site for cleave
with 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
recombinants —
Alkaline 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
G4 Ligation, transformation and analysis of
recombinants —
Transformation
• 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.
G4 Ligation, transformation and analysis of
recombinants —
Selection
G4 Ligation, transformation and analysis of
recombinants —
Transformation efficiency
• 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
recombinants —
Screening 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
recombinants —
Growth 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.
G4 Ligation, transformation and analysis of
recombinants —
Gel 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
recombinants —
Fragment 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 questions
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'phosphate and 5'-OH.
C requires NADH.
D joins single-stranded DNA.
5. In agarose gel electrophoresis
.
A DNA migrates towards the negative electrode.
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
the DNA.
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