An Introduction to Cloning and

Recombinant DNA
Prof. Arnaldo Ferreira
13.1 What Are Clones?

 Clones
• Genetically identical molecules, cells, or
organisms all derived from a single ancestor

 Cloning
• The production of identical copies of molecules,
cells, or organisms from a single ancestor
Cloning Higher Plants and Animals

 Development of methods for cloning higher

plants and animals represents a significant
advance in genetic technology
• Improving crops
• Producing domestic animals
Plants Can Be Cloned
from Single Cells

 1950s: Charles Steward grew individual carrot

cells in the laboratory by using special nutrients

 Single cells grew and divided to form a ball of

undifferentiated cells (callus)

 Calluses transferred to a different medium grew

into full-size carrots (clones)
A Cloned Plant

 Single cells grow and divide to form a callus

Animals Can Be Cloned
by Several Methods

 Embryo splitting
• After in vitro fertilization, early embryonic cells are
divided and grown into clones

 Nuclear transfer (cell fusion)

• Enucleated eggs are fused with embryonic or
adult cells and grown into clones
• Dolly the sheep
Why is DNA Cloning Important?

 DNA clones are used to find genes, map them,

and transfer them between species

 Cloning technology is used to find carriers of

genetic disorders, perform gene therapy, and
create disease-resistant plants
Keep In Mind

 Cloned plants and animals are used in research,

agriculture, and medicine
13.2 Cloning Genes
Is a Multistep Process

 Technology was developed to clone segments of

DNA molecules, based on enzymes (restriction
endonucleases) that recognize and cut DNA at
specific nucleotide sequences
Recombinant DNA Technology

 Recombinant DNA technology

• Techniques in which DNA fragments are linked to
self-replicating vectors to create recombinant
DNA molecules which are replicated in a host cell
What’s Needed to Clone DNA?

 A way to cut DNA at specific sites

 A carrier molecule to hold DNA for cloning

 A place where the DNA can be copied (cloned)

DNA Can Be Cut at Specific Sites
Using Restriction Enzymes

 Bacteria produce restriction enzymes to protect

themselves from viral infections

 Restriction enzymes
• Bacterial enzymes that cut DNA at specific sites
Vectors are Carriers of DNA to be Cloned

 Linking DNA segments produced by restriction-

enzymes with vectors (plasmids or engineered
viral chromosomes) produces recombinant DNA

 Vectors
• Self-replicating DNA molecules used to transfer
foreign DNA segments between host cells
Cloning Recombinant DNA

 Recombinant DNA molecules are transferred

into host cells; cloned copies are produced as
the host cells grow and divide

 Most common host cell: the bacterium E. coli

 Cloned DNA molecules can be recovered from

the host cells and purified for further use
E. coli

 The recognition and cutting site for EcoRI

Plasmids

 Plasmids used as vectors for cloning DNA

Steps in the Process of Cloning

 DNA is cut with a restriction enzyme

• Fragments produced end in specific sequences

 Fragments are mixed with vector molecules cut

by the same enzyme
• DNA ligase joins recombinant DNA molecules
Steps in the Process of Cloning

 Plasmid vectors with inserted DNA fragments

are transferred into bacterial cells
• Recombinant plasmids replicate and produce
many clones of the recombinant DNA molecule

 Colonies carrying cloned recombinant DNA

molecules are identified, collected, and grown
• Host cells are broken open and recombinant
plasmids are extracted
Cloning Bacteria on Petri Plates

 Each colony is a clone, descended from a single

cell
Identifying Colonies
With Recombinant DNA

 Plasmid pBR322 has been engineered to carry

two antibiotic-resistance genes with restriction
sites, one for tetracycline, one for ampicillin

 Colonies with human DNA inserted into the

tetracycline gene will not grow on tetracycline
plates, but will grow on ampicillin plates
Antibiotic-Resistance Genes
on Plasmid pBR322

 Two antibiotic-resistance genes, one for

tetracycline, one for ampicillin
Keep In Mind

 Cloning DNA uses techniques of biochemistry,

genetics, and molecular biology
13.3 Cloned Libraries

 A collection of cloned DNA sequences from one

source is a library
• Genomic library
• Chromosomal library
• Expressed sequence library

 Libraries are resources for gene studies

• Stored on yeast artificial chromosomes (YACs)
Storing a Genomic Library

 Genomic library
• A collection of clones that contains all the genetic
information in an individual

 Yeast artificial chromosome (YAC)

• Cloning vector with telomeres and a centromere
• Carries DNA fragments up to 1 million bases long
• Uses the eukaryote yeast as a host cell
Genetics in Society
Asilomar: Scientists Get Involved

 An international conference was held at

Asilomar, California, to consider the possible
dangers of recombinant DNA technology
• In 1976, guidelines were set in place for
experiments using recombinant bacteria
• New guidelines were published in 1982
• No experiments are currently prohibited
13.4 Finding a Specific Clone in a Library

 Clones for specific genes can be recovered from

a library by using probes to screen the library

 Probe
• A labeled nucleic acid used to identify a
complementary region in a clone or genome
Keep In Mind

 Finding a specific gene in a cloned library

requires a molecular probe
13.5 A Revolution in Cloning:
The Polymerase Chain Reaction

 Polymerase chain reaction (PCR)

• A method for amplifying DNA segments using
cycles of denaturation, annealing to primers, and
DNA polymerase-directed DNA synthesis

 PCR copies a DNA molecule without restriction

enzymes, vectors, or host cells
• Faster and easier than conventional cloning
First Step in PCR: Denaturation

1. DNA is heated to break the hydrogen bonds

between the two polynucleotide strands
• Two single-stranded DNA molecules serve as
templates
Second Step in PCR: Annealing

2. Short nucleotide sequences (primers for DNA

replication) are mixed with the DNA and bind to
complementary regions on single-stranded DNA
• Takes place at lower temperature
• Primers are 20-30 nucleotides long, synthesized
in the laboratory
Third Step in PCR: DNA Synthesis

3. The enzyme Taq polymerase is added to

synthesize a complementary DNA strand
• Taq is a DNA polymerase from a bacterium found
in hot springs

 These three steps make up one PCR cycle

Many Uses for PCR

 DNA to be amplified by PCR does not have to

be purified and can be present in small amounts
• Used in clinical diagnosis, forensics, conservation
• Samples can be small or old (insects in amber)
Keep In Mind

 The polymerase chain reaction (PCR) copies

DNA without cloning
13.6 Analyzing Cloned Sequences

 Cloned sequences are characterized in several

ways, including Southern blotting and DNA
sequencing

 Southern blot
• A method for transferring DNA fragments from a
gel to a membrane filter, developed by Edwin
Southern for use in hybridization experiments
Southern Blotting Can Be
Used to Analyze Cloned Sequences

 Southern blotting uses gel electrophoresis to

separate DNA fragments
• DNA fragments migrate through the gel from a
negative pole to a positive pole
• Small fragments move faster than large
fragments

 Radioactive probes identify blotted bands

DNA Sequencing
Can Be Done for an Entire Genome

 DNA sequencing
• A technique for determining the nucleotide
sequence of a fragment of DNA
• Basic method used in genome projects

 There are several ways to sequence DNA

Automated Sanger Method

 DNA is separated into strands

 DNA polymerase, a primer, and four kinds of

altered nucleotides are added
• Each nucleotide fluoresces a different color

 When an altered nucleotide is added, synthesis

stops; strands of every length accumulate
• Fragments are separated by length and scanned
with a laser that reveals the fluorescent tag
Genetic Journeys: DNA Sequencing

 In 1977, Fred Sanger sequenced the 5,400

nucleotides in the genome of a virus

 Automated methods allowed the human genome

(3.2 billion nucleotides) to be sequenced

 DNA sequencing is one of the basic methods in

recombinant DNA technology