cDNA,

cDNA Library and

Cloning
• At the end of this section, you will be in a position
to:
• Define cloning, restriction endonuclease, cDNA
and cDNA library and other related terms
• Understand the purpose of cloning in medicine
• Appreciate the applications of genetic cloning
• Understand the tools used for cloning
• Explain how cDNA libraries are created
WHAT IS CLONING?

 Cloning is the creation of an exact genetic replica

of a small segment of DNA, a cell or a whole
organism.

 Identical twins are an example of human clones

that are created naturally.
• Recombinant DNA is a form of artificial DNA which
is engineered through the combination or insertion of
one or more DNA strands, there by combining DNA
sequences which would not normally occur together

• Cloning is the process of creating an identical copy of

something.

• To clone something is to make a genetically exact

copy of it

• DNA cloning is the process of making multiple,

identical copies of a particular piece of DNA
 There are three types of cloning

(1) Recombinant DNA technology or DNA cloning,

(2) Reproductive cloning, and
(3) Therapeutic cloning.
1.Recombinant DNA Technology or
DNA Cloning

 The terms:
-“Recombinant DNA technology,"
-"DNA cloning,"
-“Molecular cloning,"or
-“Gene cloning"

 all refer to the same process:

 APPLICATIONS OF GENETIC ENGINEERING
i. Industrial and Biotechnology products e.g
manufacture of Taq pol

ii. Medical products e.g vaccines, insulin

iii. Agriculture and environment e.g plant

resistant to pesticides, GMOs

iv. Gene therapy

v. Basic Research
 Recombinant DNA Technology in the
Synthesis of Human Insulin

 Chemically, insulin is a small, simple protein.

 It consists of 51 amino acid, 153 nitrogen bases (63 in

the A chain and 90 in the B chain).
-30 of which constitute one polypeptide chain,
-and 21 of which comprise a second chain.

 The two chains are linked by disulfide bonds

 The coding sequence for insulin is found in the DNA at

the top of the short arm of the 11 chromosome.
Recombinant protein vaccines
 Recombinant proteins are produced by
recombinant DNA segments which in turn produce
proteins that serve as the immunogens.

 Hepatitis B virus; viral surface antigen produced in

yeast cells proved effective in clinical trials and
later produced the first licensed recombinant
human vaccine.

 Various recombinant proteins for HIV/AIDS are

at various stages of trials with low success.

 Several recombinant protein vaccines for malaria

are still in trials.
Restriction Endonuclease

 Restriction endonuclease [Restriction enzymes] are the

special group of bacterial enzymes that cleave double
stranded DNA at specific nucleotide sequence called
restriction site

 Also known as Molecular Scissors

 Restriction endonuclease cleaves double stranded DNA

into smaller and more manageable fragments

 These segments are called restriction fragments

 Recognize short stretches of DNA generally 4 – 6 base

pairs, that contain specific nucleotide sequences -
palindromes
 Palindromes exhibit two fold rotational symmetry
[1800 rotation].

 This means, within a short region of the double

helix DNA, if one strand is read 5’-3’ direction, the
sequence is identical to that of its complimentary
strand also read in 5’-3’.

 Restriction enzyme is named according to the

organism from which it was first isolated.
 Palindromes exhibit two fold rotational symmetry

 This means, within a short region of the double

helix DNA, if one strand is read 5’-3’ direction, the
sequence is identical to that of its complimentary
strand also read in 5’-3’.

 MADAM; ROTATOR; NURSESRUN; CIVIC

• Restriction enzymes are present in
bacteria

• Bacteria use these as defense against DNA

from bacteriophage

• DNA is cut between the 3′ hydroxyl group

of one nucleotide and the 5′ phosphate
group of the next - restriction digestion.
• Restriction enzymes do not cut bacteria’s
own DNA because the recognition
sequences are modified

• Methylases add methyl groups after

replication; makes sequence unrecognizable
by restriction enzyme
 Restriction enzymes cleave DNA so as to produce a
3’-OH group on one end and a 5’-phosphate group
on the other.

 Some restriction endonuclease, such as TaqI, form

staggered cuts that produce “sticky” or “cohesive”
ends.

 That is the resulting DNA fragment have single

stranded sequences that are complimentary to
each other.

 Other restriction endonucleases, such as HaeIII,

produces fragment that have “blunt” ends that are
double stranded and therefore do not form
hydrogen bonds with each other.
 Palindrome, Restriction Enzyme, Sticky Ends
Arber, Nathans, Smith (1978)

CIVIC, Madam Sticky Ends

(Cohesive Ends)

GAATTC G AATTC
GAATTC AATTC G

EcoRI
G AATTC
G AATTC
Get An Apple To The Class
Juang RH (2004) BCbasics
 A restriction endonuclease that recognizes a
specific four-base-pair sequence produces many
cuts in the DNA molecule.

 While the restriction endonuclease requiring a

unique sequence of six base pairs produces fewer
cuts and, hence longer pieces.

 Hundreds of these enzymes, having different

cleavage specificities are commercially available as
analytic reagents.
 Using the enzyme DNA
ligase, sticky ends of a
DNA fragment of
interest can be
covalently joined with
DNA fragment that
have sticky ends also
produced by cleavage
with the same
restriction
endonuclease.
 The Specific Cutting and Ligation of DNA
GAATTC GAATTC
CTTAAG CTTAAG

EcoRI
G AATTC G AATTC
CTTAA G CTTAA G

G G AATTC AATTC
CTTAA CTTAA G G
EcoRI sticky end EcoRI sticky end
DNA Ligase
G AATTC
CTTAA G
Juang RH (2004) BCbasics
 Nomenclature for RE
 Restriction enzyme is named according
to the organism from which it was first
isolated

 The first letter of the name is from

the genus of the bacterium.

 The next two letters are from the

name of the species.
 An additional subscript letter indicates the type
or strain, and a final number is appended to
indicate the order in which the enzyme was
discovered in that particular organism

 E.g, HaeIII is the third restriction endonuclease

isolated from bacterium Haemophilus aegyptius.
Restriction enzymes

13
Vectors
 A vector is a molecule of DNA to which the fragment
of DNA to be cloned is joined.

 Essentially, properties of a vector include:

1. It must be capable of autonomous replication within the host

cell;

2. It must contain at least one specific nucleotide sequence

recognized by a restriction endonuclease, and

3. It must carry at least one gene that confers ability to select

for the vector [e.g, antibiotic resistance gene

4. It must contain an Origin of replication (Ori)

5. Small size in comparison with host’s chromosomes

Plasmids have all these characteristics

 Plasmids are small, many have only one

restriction site

 Genes for antibiotic resistance can be

used as reporter genes

 And they have an origin of replication and

 can replicate independently

 Commonly used vectors include Plasmids and
bacteria and animal virus.

 Other vectors that can more efficiently

accommodate large DNA segments, or express the
passenger genes in different cell types are in use.

 Bacteriophage, artificial constructs such as

cosmids, bacterial artificial chromosome [BAC] and
Yeast artificial chromosome [YAC] are currently in
wide use as cloning vectors.
Plasmids
 Prokaryotic organisms contain single, large, circular
chromosome.

 Plasmids are small, circular, extra-chromosomal

fragments of DNA present in some bacteria.

 Plasmid DNA undergoes replication that may or

may not be synchronized with the chromosomal
division.

 Plasmids may carry genes that convey antiobitic

resistance to the host bacterium, and may
facilitate the transfer of genetic information from
one bacterium to to another.
 A plasmid can transfer genetic material to another
bacterium, allowing it to express the transmitted
gene(s)

 Plasmids can be readily isolated from bacterial cells,

their circular DNA cleaved at specific sites by
restriction endonuclease and foreign DNA inserted.

 The hybrid [recombinant] plasmid can be re-

inserted into a bacterium in order to:

- Clone a plasmid containing the foreign DNA segment

[to make many copies of a hybrid plasmid]

- Express the gene and produce the protein coded for

by the gene.
 Plasmids are used in genetic engineering because
they are self replicating circular DNA.

 They are vectors, used to transfer genes from one

organism to another and,

 typically contain a genetic marker conferring a

phenotype that can be selected for or against.

 Most also contain a polylinker or multiple cloning

site (MCS),

 which is a short region containing several commonly

used restriction sites allowing the easy insertion
of DNA fragments at this location.
 Cloning vector plasmids
• Modified plasmids, called cloning vectors
• Are used by molecular biologists to isolate Large
quantities of a given DNA fragment

• Plasmids used for cloning share three properties

 Unique restriction site

 Antibiotic resistance

 Origin of replication
 Plasmid Cloning vector
pBR322
• pBR322 is a plasmid and was one of the
first widely used E. coli cloning vectors.
• Created in 1977 in the laboratory
of Herbert Boyer at the University of
California, San Francisco,
• it was named after the postdoctoral
researchers who constructed it.
• The p stands for "plasmid," and BR for
"Bolivar" and "Rodriguez."
 Plasmid Cloning vector pBR322
• pBR322 is 4361 base pairs in length and
contains;
– Resistance for 2 antibiotics
• Tetracycline
• Ampicillin
– Origin of replication between the 2
resistance genes
– Only 1 site for several restriction
enzymes
 Plasmid vector elements
1. Origin of replication:

•This is a DNA element that allows the plasmid to be

replicated and duplicated in bacteria.
•Each time the bacterium divides, the plasmid also
needs to divide and go with the daughter cells.
•If a plasmid cannot replicate in bacteria, then it will be
lost.
2. Antibiotic resistance gene:

•This allows for the presence of the plasmid to be

selectively maintained in a given strain of bacteria

Lab bacterial strains are sensitive to antibiotics.

•When grown on plates with antibiotics, they die.
•The presence of a plasmid with the antibiotics resistance
gene allows these lab strains to grow on plates with the
antibiotic.
•You are therefore selecting for bacterial colonies with
the Plasmid
3. Unique restriction sites:

For cloning, the plasmid needs to be linearized.

Most cloning vectors have unique restriction sites.
If the plasmid contains more than one site for a given
restriction enzyme, this results in fragmentation of the
plasmid

4. Multiple cloning site / polylinker region

Is a unique sequence site that can be recognized by

Restriction enzymes and and cleaved to open the plasmid to
create a space where a new insert (gene/DNA segment to be
cloned) is ligated
Cloning using pBR322
• If new DNA is inserted at that PstI
restriction site, it inactivates the gene
for ampicillin resistance

• Plasmid then has gene for tetracyclin

resistance, but not for ampicillin.

• This can be used to select for host cells

with new DNA.
Cloning using pBR322
• Clone a foreign DNA into
the PstI site of pBR322
• Cut the vector to
generate the sticky ends
• Cut foreign DNA with
PstI also – compatible ends
• Combine vector and
foreign DNA with DNA
ligase to seal sticky ends
• Now transform the
plasmid into E. coli
4-46
 Cloning using pBR322
The cleaved plasmid
DNA preparation is
treated with enzyme
alkaline phosphatase
to remove the 5’
phosphate groups from
the linearized plasmid
DNA.
cDNA

 In genetics, complementary DNA (cDNA) is DNA

synthesized from a mature mRNA template.

 Eukaryotic DNA has introns, these have to be

removed and exons spliced.

 This spliced DNA is called complementary DNA.

Synthesis
 cDNA is most often synthesized from mature
(fully spliced) mRNA using the enzyme reverse
transcriptase.

 This enzyme operates on a single strand of mRNA,

 generating its complementary DNA based on the

pairing of RNA base pairs (A, U, G, C) to their
DNA complements (T, A, C, G).
 Intron and Exon in Eukaryotic Cells

exon exon exon

promotor intron intron DNA
3’ 5’
start codon stop codon
Transcription
5’ 3’ mRNA
Processing
cap

Splicing poly A
tail
Intron deleted

mature mRNA To cytoplasm

Take place in nucleus
Juang RH (2004) BCbasics
 cDNA Is Reverse Transcribed from mRNA
Baltimore, Dulbecco, Temin (1975)

mature mRNA
5’ 3’
poly A tail
Reverse
transcription TTTT
3’ 5’
RNA hydrolysis

3’ 5’
DNA polymerase

3’ CCC 5’
5’ GGG 3’
Juang RH (2004) BCbasics
 Two Libraries ： cDNA Library vs Genomic Library

Genes in expression Total Gene

mRNA Chromosomal DNA

Reverse transcription
Restriction digestion

Complete
cDNA gene Gene fragments

Smaller Larger Vector:

Vector: Plasmid Library Library Plasmid or phage
Juang RH (2004) BCbasics
 To obtain eukaryotic cDNA whose introns
have been spliced:

 A eukaryotic cell transcribes the DNA (from

genes) into RNA (pre-mRNA).

 The same cell processes the pre-mRNA strands

by removing out introns, splicing the exons and
adding a poly-A tail and 5’ Methyl-Guanine cap.

 This mixture of mature mRNA strands are

extracted from the cell.
4. A poly-T oligonucleotide primer is hybridized onto
the poly-A tail of the mature mRNA template
(Reverse transcriptase requires this double-
stranded segment as a primer to start its
operation).

5. Reverse transcriptase is added, along with

deoxynucleoside triphosphates (A, T, G, C)

• The reverse transcriptase scans the mature mRNA

and synthesizes a sequence of DNA that
complements the mRNA template.

• This strand of DNA is complementary DNA

[cDNA]
cDNA library

 cDNA library refers to a complete, or nearly

complete, set of cDNA of all the mRNAs contained
within a cell or organism.

 Because working with mRNA is difficult (as mRNA

is unstable and is easily degraded by RNases which
can be found even on the skin),

 researchers use an enzyme called reverse

transcriptase which will produce a DNA copy of
each mRNA strand.

 Referred to as cDNA these reverse transcribed

mRNAs are collectively known as the library.
 cDNA library construction

 cDNA libraries are prepared from total or

enriched Poly(A) + single stranded mRNA that is
converted into a double-stranded DNA copy of the
message using the enzyme reverse transcriptase.

 The cDNA fragments can be inserted into an

appropriate plasmid, phage, or cosmid vector for
maintenance and cloning.

 The population of recombinant vectors will

represent the entire set of expressed genes in the
cell from which the RNA was isolated.
2. Reproductive Cloning

 Reproductive cloning is a technology used to

generate an animal that has the same nuclear DNA
as another currently or previously existing animal.

 It’s done in a process called "somatic cell nuclear

transfer" (SCNT),

 genetic material is transferred from the nucleus

of a donor adult cell to an egg whose nucleus, and
thus its genetic material, has been removed.
 The reconstructed egg containing the DNA from a
donor cell must be treated with chemicals or
electric current in order to stimulate cell division.

 Once the cloned embryo reaches a suitable stage,

it is transferred to the uterus of a female host
where it continues to develop until birth.
Benefits and limitations

 Reproductive cloning can have many uses.

 If the low success rates and issues of safety could

be improved, the technology can be used to mass-
produce animals with special qualities,

 such as animals that are important agriculturally or

are able to produce helpful drugs for human use.
 SCNT has environmental uses in that it can be
used to repopulate endangered species, as has
been shown with the wild ox, the gaur.

 Some supporters of human reproductive cloning

see it as a way of overcoming male infertility,
where other methods of assisted reproduction
have failed.
Ethical considerations

 Human reproductive cloning has raised many

concerns, not the least of which are safety
considerations.

 More than 90% of off spring from cloning are not

viable:

 only 1 or 2 offspring are viable for every 100

cloning attempts.
 In addition to this low success rate, cloned animals
have higher rates of cancer and infection, and a
range of other disabilities and conditions.

 Also while the animal may seem healthy at birth,

problems often have appeared later in the life of
the resulting clone,

 so that mature clones have often undergone

sudden, unforeseen and unexplainable deaths.
 reproductive cloning of humans is viewed
differently.

 Intellectual and emotional development is essential

for human growth and health.

 It is also important to realize that a person is

much more than a product of their genes.
 A person is a product of their environment as well
as their genetic make-up.

 Even identical twins have subtle differences

between them

 The concepts of parenthood, “family” and views of

social responsibilities are all challenged by human
reproductive cloning.
3.Therapeutic cloning

 Therapeutic cloning, also called "embryo cloning,"

is the production of human embryos for use in
research.

 The goal of this process is not to create cloned

human beings,

 but rather to harvest stem cells that can be used

to study human development and to treat
disease.
 Stem cells are extracted from the egg after it has
divided for 5 days.

 The egg at this stage of development is called a

blastocyst.

 The extraction process destroys the embryo,

which raises a variety of ethical concerns.
 Embryonic stem cells still have all the genes
needed for the function of the body’s cells able to
be turned on and so can be differentiated into any
type of body cell.

 So, to generate cultures of specific types of

differentiated cells (e.g. heart muscle cells, blood
cells, or nerve cells) scientists try to control the
differentiation of embryonic stem cells.

 They change the chemical composition of the

culture medium, alter the surface of the culture
dish, or modify the cells by inserting specific
genes.