An Introduction to Cloning and An Introduction to Cloning and Recombinant DNARecombinant DNA

Prof. Arnaldo FerreiraProf. 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 SitesUsing 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