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Lecture 6:

Gene Cloning and Analysis

Outline
1.
2.
3.
4.
5.
6.

What is gene cloning


Overview of the procedures
Extraction and purification of nucleic acids
Plasmid vectors
Ligation and transformation
Vector based on the lambda bacteriophage,
cosmids and supervectors

What Is Cloning?
Using asexual reproduction to obtain organisms that are
genetically identical to one another, and to the parent
Clones produced are only identical genetically; the actual
appearance and behavior of the clones will be influenced by
other factors such as their environment
Example 1:
to propagate plants by taking cuttings, and produce a number
of plants that are identical to the parent. These new plants are
clones

Example 2:
Purifying a bacterial strain by picking a single colony for
inoculation a series of fresh cultures is also a form of cloning

What is Gene Cloning?


A fragment of DNA, containing the gene to be cloned, is inserted into
a circular DNA molecule called a vector, to produce a chimera or
recombinant DNA molecule
The vector acts as a vehicle that transports the gene into a host cell,
which is usually a bacterium
Within the host cell the vector multiplies, producing numerous
identical copies not only of itself but also the gene that it carries
When the host cell divides, copies of the recombinant DNA molecule
are passed to the progeny and further vector replication takes place
After a large number of cell divisions, a colony, or clone, of identical
host cells is produced. Each cell in the clone contains multiple copies
of the recombinant DNA molecule
Cloning can provide a pure sample of an individual gene, separated
from all the other genes that it normally shares the cell with

Overview of the Procedures

Extraction and Purification of Nucleic Acids


The 1st step for the proceduresextract the DNA
or RNA from the cell and to purify it by
separating it from other cellular components
Most commonly used methods of purifying and
fractionating nucleic acids are as follow:
1. Breaking up cells and tissues
2. Purification and concentrating of the nucleic
acids
3. Detection and quantification of nucleic acids

1. Breaking up cells and tissues


Separate the cell from the growth medium (e.g. by
centrifugation)
Cell need to be lysedto release their components by
using a combination of EDTA, lysozyme and a
detergent such as SDS
EDTAeliminates divalent cations and thus
destabilizes the outer membrane, allowing the
lysozyme to access the cell wall structure
Lysozymedigest the polimer that form the rigid cell
wall
Detergentto solubilize the membrane lipids,
releasing the cell contents

What do you get after the lysis?


A crude extracta complex miture of DNA,
RNA, proteins, lipids and carbohydrates
Next stepto separate the desired nucleic acids
from other components
Removal of proteinsextraction with a mixture
of liquefied phenol and chloroform or digestion
with a proteolytic enzyme such as proteinase K

2. Purification and concentrating


of the nucleic acids
Ethanol precipitation

Column purification

3. Detection and quantification of


nucleic acids
Estimate the concentration by measuring the
absorbance in an UV spectrophotometer at
260nm
260:280 absorbance ratio between 1.75 and 2=
pure (acceptable)
*UV absorbance only gives the estimate of
concentration, not the integrity of the DNA
Gel electrophoresisfor both detecting and
quantitating nucleic acids

Gel electrophoresis
Ethidium bromide agarose gel

Overview of the Procedures

Plasmid as cloning vector:


Plasmid DNA can be transferred from one bacterial
cell to another during conjugation
Transport requires a mobility protein (encoded by
the plasmid-borne mob gene) and a specific site
(nic) on the plasmid
The mob protein nicks the plasmid at nic and then
attaches to the nicked strand to conduct the
plasmid through a mating channel (pilus) into a
recipient cell
Cloning vectors lack both the mob gene and the nic
site and thus cannot be mobilized for transport

mob
gene

Plasmid

mob
protein
nic
site

Plasmid Vectors
Gene cloning requires specialized tools and
techniques
The Vehicles
Transport the gene into the host cell and is
responsible for its replication
Must be capable of entering a host cell, replication
to produce multiple copies of itself

Basic features of plasmids:


Circular molecules of DNA that lead an independent
existence in the bacterial cell as extrachromosomal
DNA molecules
Usually circular, double-stranded and supercoiled
Occur widely in nature, and are found in most
bacterial species
Always carry one or more genes,exp: antibiotics
resistance (ampicillin, tetracycline, kanamycin.)
used as a selectable marker

Origin of replication: able to multiply within the


cell independently
Smaller plasmids use of the host cells own DNA
replicative enzymes, larger ones carry genes that
are specific for plasmid replication
Plasmid size range from about 1.0 kb to 250 kb
Copy number: present in the cell as one (large
plasmids) or as many as 50 or more for smaller
plasmid

Based on small bacterial plasmids (pUC18/pUC19,


pBluScripts) avoid problems such as DNA breakdown

High transformation efficiency

Convenient selectable markers (antibiotic or lacZ gene)

The ability to clone reasonably wide range of DNA


inserts (0.1 kb 10 kb)

High copy number of 500-1000 copy per cell

Do not integrate into the host genome

Cloning sites/multiple cloning site/polylinker cloning


sites

Unique restriction enzyme on MCS only one site in the


vector

Overview of the Procedures

Ligation and Transformation


Cloning serves two main purposes:
Allow a large number of recombinant DNA
molecules to be produced from a limited
amount of starting material
Allow the purification of recombinant DNA
molecules

The ligation mixture may contain:

Unligated molecules will be degraded by the


host enzymes, if go into the host cell
Each host cell can only taken up one DNA
molecule
Each cell gives rise to a single colony, so each of
the resulting clones consists of cells that all
contain the same molecule.

Preparation of competent E. coli cells for transformation:

1. Usually 50 mM calcium chloride is used.


2. CaCl2 causes the DNA to precipitate on
membrane cells
3. CaCl2 responsible for change in the cell wall
improves DNA binding
4. Movement of DNA into competent cells is
stimulated by briefly raising the temperature to
42C

Transformation protocols:
1. Preincubation:

Cells are suspended in a solution of cations,


CaCl2, at 0oC.

2. Incubation
. DNA is added, and the cell suspension is further
incubated at 0oC. The cations are thought to
neutralize negatively charged phosphates in the
DNA and the cell membrane

3. Heat shock

The cell/DNA suspension is briefly incubated at


42oC and then returned to 0oC
The rapid temperature change creates a thermal
imbalance on either side of the E. coli membrane
Sweeps plasmids into the cell.

4. Recovery

LB broth is added to the DNA/cell suspension and


incubated at 37oC before plating on selective media
Transformed cells recover from the treatment,
begin to express the antibiotic-resistance protein

Selection for transformed cells:


A selectable marker - a gene that provides a
transformed cell with a new characteristic, not
possessed by non-transformant
The resistance gene on the plasmid must be
expressed, so that the enzyme that detoxifies the
antibiotic is synthesized
Expression of the resistance gene begins
immediatelybefore the cell contains enough of
the enzyme to be able to withstand the toxic
effects of the antibiotic

Transformed bacteria first placed in a small


volume of liquid medium, absence of antibiotic,
incubated for a short time
So that when the cells are plated out and
encounter the antibiotic, they will already have
synthesized sufficient resistance enzymes to be
able to survive

Selection based on pBR322:

If a new fragment of
DNA is ligated into one
of these sites, the
antibiotic-resistance
genes become altered

The plasmid loses the


ability to confer either
ampicillin or
tetracycline resistance
on the host

This is called
insertional
inactivation of the
selectable marker

The resistance properties of colonies are tested by transferring cells


from agar containing one antibiotic onto agar containing the second
antibiotic

If the BamHI site has been used then recombinants will be


ampicillin resistant but tetracycline sensitive

After transformation, cells are plated onto ampicillin agar

All cells contain a pBR322 plasmid, whether recombinant or not,


divide and produce a colony

The colonies are then transferred onto tetracycline agar by replica


plating

Some colonies do not grow on the tetracycline agar because their


cells contain recombinant pBR322 molecules with a disrupted
tetracycline-resistance gene

These colonies contain the cloned gene. These colonies can be


recovered by returning to ampicillin plate

Identification of recombinants
1. Antibiotic resistance selectable marker
2. lacZ selectable marker
3. Colony hybridization
4. Colony-PCR

lacZ selectable marker (blue-white screening):

1. lacZ is a portion of bacterial gene, which codes for


part of the enzyme -galactosidase
-galactosidase involved in the breakdown of lactose
to glucose plus galactose
3. It is normally coded by the gene lacZ, resides on the
E. coli chromosome
4. Some strains of E. coli have a modified lacZ gene,
which lacks the segment referred to as lacZ and
coding for the -peptide portion of -galactosidase

5. These mutants can synthesize the enzyme only when


they harbour a plasmid, such as pUC18/19/8, that
carries the missing lacZ segment of the gene.
6. The proteins specified by the gene segments on the
plasmid and on the chromosome are able to combine
to produce a functional -galactosidase enzyme.
7. The lacZ gene contains a cluster of unique
restriction sites; insertion of new DNA into any one
of these sites results in insertional inactivation of the
gene and hence loss of -galactosidase activity.

8. Screening using lactose analogue called X-gal (5bromo-4-chloro-3-indoly--D-galactopyranoside)


which is broken down by -galactosidase to a
product that is coloured deep blue
9. Inducer of the enzyme such as isopropylthiogalctoside, IPTG is used
10.Non-recombinant colonies, will be coloured blue
11. Recombinants colonies, will be in white colour

End of Lecture 6
Thank You