BTH 202: Principles of Plant Biotechnology

PLANT DNA CLONING

DR. SULAIMAN MOHAMMED

 OVERVIEW: THE DNA TOOLBOX
ú Sequencing of the genomes of more than 7,000
species was under way.
ú DNA sequencing has depended on advances in
technology, starting with making recombinant DNA
(rDNA).
ú Methods for making recombinant DNA are central to
genetic engineering, i.e. the direct manipulation of
genes for practical purposes.
ú rDNA is possible because DNA molecules from all
organisms share the same chemical structure. They
differ only in the nucleotide sequence.
ú rDNA is the general name for a piece of DNA that has
been created by the combination of at least two
strands.
ú rDNA technology has revolutionized biotechnology,
the manipulation of organisms or their genetic
components to make useful products.
ú An example of rDNA technology is the measurement
of gene expression of thousands of different genes.
 DNA CLONING: A Preview
§ In recombinant DNA technology; gene sequences from two
different sources are combined (cloning) in vitro into the same
DNA molecule.
§ creating sequences that would not otherwise be found in the
genome.
§ rDNA technology uses palindromic sequence and leads to the
production of sticky or blunt ends.
§ Most methods for cloning pieces of DNA in the laboratory
share general features, e.g. the use of bacteria (host: to
make multiple copies of a gene) and their plasmids (as
vehicle):
§ Plasmids are small circular DNA molecules that replicate
separately from the bacterial chromosome
§ Foreign DNA is ligated to a plasmid, and the recombinant
plasmid is introduced into a bacterial cell
Plasmid vector (for bacterial transformation)
Binary vector
§ Reproduction in the bacterial cell results in
multiplication of the plasmid including the foreign
DNA.
§ This results in the production of multiple copies of a
single gene.
§ Cloned genes are useful for making copies of a
particular gene and producing a protein product.
DNA cloning yields multiple copies of a gene
or other DNA segment

• To work directly with specific genes, scientists

prepare well-defined segments of DNA in identical
copies, then join together a process called DNA
cloning
DNA Cloning and Its Applications: A Preview
• Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use
of bacteria and their plasmids, plus foreign DNA
• Plasmids are small circular DNA molecules that
replicate separately from the bacterial chromosome
• Cloned genes are useful for making copies of a
particular gene and producing a protein product
• Gene cloning involves using bacteria to make
multiple copies of a gene
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial
cell
• Reproduction in the bacterial cell results in the
production of multiple copies of the clone gene
 Bacterium
1 Gene inserted into
Cell containing
plasmid
gene of interest

Bacterial Plasmid
chromosome Gene of
Recombinant interest DNA of
DNA (plasmid) chromosome
2 Plasmid put into
bacterial cell (“foreign” DNA)

Recombinant
bacterium
Figure 20.2b

3 Host cell grown in

culture to form a clone
of cells containing the
“cloned” gene of interest
Gene of Protein expressed from
interest gene of interest
Copies of gene Protein harvested

4 Basic research
Basic and various Basic
research applications research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth
Using Restriction Enzymes to Make Recombinant
DNA
• Bacterial restriction enzymes cut DNA molecules at
specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts,
yielding restriction fragments
• The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky
ends”
• Sticky ends can bond with complementary sticky ends
of other fragments
• DNA ligase is an enzyme that seals the bonds
between restriction fragments
Sticky ends Blunt end
Figure 20.3-3
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3 5
5 G AATTC 3
CTTAA G
5 Sticky 3
3 5
end
5
AATTC 3
G G
CTTAA
2 DNA fragment added 3
from another molecule 5
cut by same enzyme.
Base pairing occurs.

5 3 5 3 5 3
G AATT C G AATT C
C TTAA G C TTAA G
3 53 5 3 5
One possible combination
3 DNA ligase
seals strands
5 3

3 Recombinant DNA molecule 5

Cloning a Plant Gene in a Bacterial Plasmid
• In gene cloning, the original plasmid is called a
cloning vector
• A cloning vector is a DNA molecule that can carry
foreign DNA into a host plant cell and replicate there
• Several steps are required to clone the plant gene in
a bacterial plasmid
– The plant genomic DNA and a bacterial plasmid are
isolated
– Both are cut with the same restriction enzyme
– The fragments are mixed, and DNA ligase is added
to bond the fragment sticky / blunt ends
‒ Some recombinant plasmids now contain plant
DNA
‒ The DNA mixture (recombinant plasmids) is
added to bacteria to accept it
‒ The bacteria are plated on a type of agar that
selects for the bacteria with recombinant
plasmids
‒ This results in the cloning of many plant DNA
fragments, including the Hd3a rice florigen
TECHNIQUE Plant cell
Bacterial plasmid
ampR gene lacZ gene
Restriction
site

Sticky
ends Gene of
interest
Plant DNA
fragments

Recombinant plasmids Non-recombinant plasmid

Bacteria carrying
plasmids
 Bacteria carrying
diverse plasmids

RESULTS

Colony carrying
Colony carrying non- recombinant
recombinant plasmid plasmid
with intact lacZ gene with disrupted
lacZ gene

One of many
bacterial
clones
 Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is
the collection of recombinant vector clones
produced by cloning DNA fragments from an entire
genome
• A genomic library that is made using plant genome
is stored as a collection of plant clones
 Foreign genome

Figure 20.5Cutwith restriction enzymes into either

small or large Bacterial Artificial
fragments fragments Chromosome (BAC)
Large
insert
with
many
genes

Recombinant (b) BAC clone

plasmids

Plasmid
clone

(a) Plasmid library (c) Storing genome libraries

• A bacterial artificial chromosome (BAC) is a
large plasmid that has been trimmed down and
can carry a large DNA insert
• BACs are another type of vector used in DNA
library construction
• A complementary DNA (cDNA) library is made
by cloning DNA made in vitro by reverse
transcription of all the mRNA produced by a
particular cell
• A cDNA library represents only part of the
genome—only the subset of genes (coding region)
transcribed into mRNA in the original cells
 DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand

5 A A A A A A 3
3 T T T T T 5

5 3
3 5
DNA
polymerase

5 3
3 5
cDNA
 Selection and Screening
q Selection; grow up the cells on antibiotic plate
containing chromogenic agent.
q Screening; rDNA molecule is smaller and can
be easily separated based on the size by
plasmid extraction method.
– Colony PCR, then run the amplicon on gel.
– Bacterial cells are broken-open to release the rDNA.
– The plasmid must be purified before cutting with REs
and running on gel.
 How to Tell that Plasmid Contain Insert

• Original vector = 3 kb
• inserted DNA = 1 kb, flanked by same RE
…Double digest the rDNA isolated from colonies (transformed).

then, run on gel : RESULTS

Vector alone (no insert) = 1 band 3 kb (unsuccessful)
Vector + insert = 2 bands; 3 kb and 1 kb (successful)
Expressing Cloned Plant Genes
• After a gene has been cloned, its protein product
can be produced in larger amounts for research
• Cloned genes can be expressed as protein in plant
cells
Question

The end