OVERVIEW:

1. Cloning : How to photocopy heredity

2. Cloning : Using a gene library

3. Cloning gene in yeast cells and mammalian host

1. CLONING : HOW TO PHOTOCOPY HEREDITY

• Cloning in biology is the process of producing populations of

genetically-identical individuals that occurs in nature when
organisms such as bacteria, insects or plant reproduce asexually.
• Cloning in biotechnology refers to processes used to create copies
of DNA fragments (molecular cloning), cells (cell cloning), or
organisms.
• Gene cloning is the technique of producing a hybrid DNA (or
recombinant) molecule in vitro which, following its introduction into a
living cell, can replicate itself and be passed from generation to
generation.
• Because the molecule into which the gene (or genes) is introduced
is able to replicate independently, it is often referred to as a replicon.
Replicon employed in gene cloning are called cloning vector and
derived from plasmids, bacteriophage, or other viruses.
• The term recombinant DNA has two meanings in genetics.
• The more specific of the two refers to a DNA molecules formed in the
laboratory by joining together DNA sequence from different sources.
• Such molecules are artificial laboratory creations and are not found in
nature.
• The term recombinant DNA is also used more loosely to refer to the
technology that is utilized to create and study these hybrid molecules
constructed from DNA obtained from different biological sources.
• Such techniques to create, clone (replicate), and analyze recombinant
DNA molecules were developed in the 1970s as a way for researcher
to isolate and study specific DNA sequences.
• The power of recombinant DNA technology is astonishing, making it
possible to identify and isolate a single gene or DNA segment of
interest from the thousand or tens of thousand present in genome.
• Subsequently, through cloning huge quantities of identical copies of
this specific DNA molecule can be produced.
• These identical copies can then be manipulated for numerous
purposes,
• Including research into the structure and organization of the DNA, or
for the commercial production of its encoded protein.
 An Overview of Recombinant DNA Technology
• Recombinant DNA technology uses methods derived from nucleic
acid biochemistry, coupled with genetics techniques originally
developed for the study of bacteria and viruses.
• This technology is a powerful tool for isolating potential unlimited
quantities of gene.
• A basic procedure for making and cloning recombinant DNA
molecules uses the following steps:
1. The DNA to be cloned is purified from cells or tissues
2. Proteins called restriction enzymes are used to generate specific
DNA fragments. These enzymes recognize and cut DNA molecules
at specific nucleotide sequences.
3. The fragments produced by restriction enzymes are joined to other
DNA molecules that serve as vector*, or carrier molecules. A vector
joined to a DNA fragment is a recombinant DNA (* A cloning vector
is a small piece of DNA into which a foreign DNA fragment can be
inserted.
 The insertion of the fragment into the cloning vector is carried out
by treating the vehicle and the foreign DNA with the same
restriction enzyme, then ligating the fragments together. There
are many types of cloning vectors. Genetically engineered
plasmids and bacteriophages (such as phage λ) are perhaps
most commonly used for this purpose).
4. As host cell replicates, the cloned DNA molecules within them are
passed to all their progeny, creating a population of host cells,
each of which carries copies of the cloned DNA sequence.
5. The cloned DNA can be covered from host cells and purified for
use in research or commercial applications.
Cloning with a plasmid vector involves cutting both plasmid and the DNA to be cloned with
the same restriction enzymes.The DNA to be cloned is spliced into the vector and transferred
to a bacteria host for replication. Bacterial cells carrying plasmids with DNA inserts can be
identify by selection of screening and then isolated. The cloned DNA is then recovered from
the bacterial host for further analysis
• Recombination occurs through two mode:
1. General (non-homologus) recombinantion
• This occur between sections of DNA which have identical, or at
least very similar, base sequences.
• Several models for general recombination have been proposed,
most of them sharing several features with the so-called Holiday
model.
• General recombination is an enzyme-mediated process which
involves the coordinated breakage and reunion of single strands of
DNA
2. Site-specific recombination
• In some cases, recombination requires the presences of specific
sites in the DNA.
• Site specific recombination is exemplified by phage lambda.
• These recombination events, which usually involve sequence of
fewer than 25 base pairs.
2. CLONING: USING A GENE LIBRARY
• In molecular biology, a library is a collection of molecules in a stable
form that represents some aspect of an organism.
• Two common types of libraries are cDNA libraries (formed from
Complementary DNA) and genomic libraries.
• The nucleotide sequences of interest are preserved as inserts to a
plasmid or the genome of a bacteriophage that has been used to
infect bacterial cells.
• DNA cloning is often used to produce DNA libraries, which are
collections of cloned DNA fragments.
 GENOMIC LIBRARY

• Genomic libraries are produced from a total DNA extracted from

nuclei and contain all of the DNA sequence of the species. Once a
genomic library of a species is available, researcher can use the
collection to isolate specific DNA sequences, such as those containing
the human insulin gene.
• A genomic library is a set of clones, packaged in the same vector, that
together represents all regions of the genome of the organism in
question.
• The number of clones that constitute a genomic library depends on:

- The size of the genome in question,

- The insert size tolerated by the particular cloning vector system.
• Genomic DNA is treated with one of two restriction enzymes that
recognize very short nucleotides sequence under conditions of low
enzyme concentration, such that only a small percentage of
susceptible sites are actually cleaved,
 cDNA LIBRARY

• A cDNA library is a collection of cloned cDNA (complementary DNA)

fragments inserted into a collection of host cells, which together
constitute some portion of the transcriptome (the set of all mesenger
RNA (mRNA) molecules, or "transcripts," produced in one or a
population of cells) of the organism.
• cDNA libraries are derived from DNA copies of RNA population. cDNA
libraries are typically produced from messenger RNAs present in a
particular cell type and thus correspond to the genes that are active in
that type of cell.

• cDNA is produced from fully transcribed mRNA found in the nucleus

and therefore contains only the expressed genes of an organism.

• A cDNA library represents all of the mRNA present in a particular tissue,

which has been converted back to a DNA template by the use of the
enzyme reverse transcriptase.
• It represents the genes that are transcribed in particular tissues under
particular physiological, developmental, or environmental conditions.

• While information in cDNA libraries is a powerful and useful tool since

gene products are easily identified, the libraries lack information about
enhancers, introns, and other regulatory elements found in a genomic
DNA library.

• cDNA libraries are useful in reverse genetics, but should not be

confused with a genomic library, as it does not represent the entire
genome, only a very small (less than 1%) portion which is being
transcribed.

• Usually a cDNA library is created when reproducing eukaryotic

genomic material, whereas genomic libraries are often created when
working with genomic target material from bacteria and viruses.
cDNA Library Construction

• cDNA is created from a mature mRNA from an eukaryotic cell with the
use of an enzyme known as reverse transcriptase. In eukaryotes, a
poly-(A) tail (consisting of a long sequence of adenine nucleotides)
distinguishes mRNA from tRNA and rRNA and can therefore be used
as a primer site for reverse transcription.

mRNA extraction:
• Firstly, the mRNA is obtained and purified from the rest of the RNAs.
Several methods exist for purifying RNA such as trizol extraction and
column purification.

• Column purification is done by using oligomeric dT nuclotide coated

resins where only the mRNA having the poly-A tail will bind.

• The rest of the RNAs are eluted out. The mRNA is eluted by using
eluting buffer and some heat to separate the mRNA strands from
oligo-dT.
 cDNA construction:
• Once mRNA is purified, oligo-dT (a short sequece of deoxy-thymine
nucleotides) is tagged as a complementary primer which binds to the
poly-A tail providing a free 3'-OH end that can be extended by reverse
transcriptase to create the complementary DNA strand.

• Now, the mRNA is removed by using a RNase enzyme leaving a single

stranded cDNA (sscDNA).

• This sscDNA is converted into a double stranded DNA with the help of
DNA polymerase. However, for DNA polymerase to synthesize a
complementary strand a free 3'-OH end is needed.

• This is provided by the sscDNA itself by generating a hair pin loop at

the 3' end by coiling on itself. The polymerase extends the 3'-OH end
and later the loop at 3' end is opened by the scissoring action of S1
nuclease. Restriction endonucleases and DNA ligase are then used to
clone the sequences into bacterial plasmids.

• The cloned bacteria are then selected , commonly through the use of
antibiotic selection. Once selected, stocks of the bacteria are created
which can later be grown and sequenced to compile the cDNA library.
 • Producing cDNA from mRNA.
Because many eukaryotic mRNA have
a polyadenylated tail (A) of variable
length at their 3’ end, a short oligo-dT
annealed to this tail serves as a primer
for the enzyme reverse transcriptase.
• Reverse transcriptase uses the mRNA
as a template to synthesize a
complementary DNA strand (cDNA)
and forms an mRNA/cDNA double-
stranded duplex.
• The mRNA is digested with the
cDNA enzyme RNAse H, producing gaps in
the RNA strand.
• The 3’ ends of the remaining RNA
serve as primers for DNA polymerase,
which synthesize a second DNA
strand.
• The result is a double-stranded cDNA
molecule that can be cloned into a
suitable vector or used directly as a
probe for library screening.
 cDNA Library uses

• cDNA libraries are commonly used when reproducing eukaryotic

genomes, as the amount of information is reduced to remove the large
numbers of non-coding regions from the library.

• cDNA libraries are most useful in reverse genetics where the additional
genomic information is of less use.

cDNA Library vs. Genomic DNA Library

• As previously mentioned, a cDNA library lacks the non-coding and

regulatory elements found in genomic DNA.

• Genomic DNA libraries provide much more detailed information

about the organism, but are much more resource-intensive to
generate and maintain.