An Introduction to Cloning and Recombinant DNA

Chapter 13

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

1950s: Charles Steward grew individual carrot cells in the laboratory by using special nutrients

Cloning is used in the paper & timber industry to produce trees of uniform size, growth rate, & disease resistance

The 2 most common methods used in cloning animals

Embryo splitting• eggs are collected and fertilized in vitro

• embryo allowed to develop to stage of 8 to 16 cells

• embryo is separated into individual cells

• cells grow in lab & are then transplanted into surrogate mothers for development

• 1894 – Hans Dreisch cloned sea urchin

The 2 most common methods used in cloning animals

Nuclear transfer / cell fusion• unfertilized eggs are collected from a donor and

enucleated

• an embryo is removed from the animal to be cloned allowed to develop to stage of 16 to 32 cells

• embryo is separated into individual cells and each is fused with an enucleated egg

• embryos are then transplanted into surrogate mothers for development

• 1986 –cloned sheep (NOT Dolly!)This technique is more difficult but can result in a

larger number of offspring

Why ~16 cell embryos?

Why ~16 cell embryos? The genetic information

in some cells is slightly modified as embryo develops (the cells may become specialized / differentiated)

How is Dolly different? In 1997 a differentiated

cell from the udder of an adult sheep was used to produce a clone animal - Dolly

Cloning Animals: Pro & Con

PRO Produce herds of

identical animals with superior wool, milk, or meat production

CON Loss of diversity Susceptibility to

disease, pests, or changing environmental conditions

Cloning of DNA / Genes

Methods for cloning DNA are referred to as recombinant DNA technology

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

What’s Needed to Clone DNA?

A way to cut DNA at specific sitesRestriction enzymes (produced by bacteria to

protect against viral infections) A carrier molecule to hold DNA for cloning

Vector is usually a bacterial plasmid (a circular DNA molecule that is self-replicating)

A place where the DNA can be copied (cloned)Host cell – most common is the bacterium E. coli

Why is a vector necessary?

Vectors are needed because linear DNA (such as human DNA) cannot be replicated within a host cell and passed on to progeny.

Vectors are self-replicating and can carry the DNA into the host cells for replication

13.2 Cloning Genes Is a Multistep Process

See page 294, 295 Cut DNA at specific sites Connect the fragments to vectors using DNA

ligase, creating recombinant DNA molecules Transfer the recombinant DNA molecule to a

host cell (where it is copied to form clones) Retrieve the cloned DNA fragment from the

vector in large quantities Turn on gene & collect & purify gene product

Recognition & cutting sequences …

Are palindromes that read the same on either DNA strand when read for 5’ to 3’ direction

This ensures that the sequence will be recognized no matter which end of the molecule the sequence is approached from

Cut, Connect, & Introduce

Cut at recognition sites using restriction enzymes

Connect to vector using DNA ligase Vector introduced or transferred to vector

Different restriction enzymes have different recognition sequences

Identifying Bacteria Colonies With Recombinant DNA

Not all vectors successfully incorporate the recombinant DNA so scientists have to have a way to identify the bacteria colonies with recombinant DNA.

Usually, the DNA is inserted in a region that codes for antibiotic resistance; if the DNA insertion is successful then the

resistance gene is nonfunctional and the colonies with human DNA inserted will not grow on growth plates that have the antibiotic as part of the growth medium

Colonies not resistant can grow on one plate but not on the plate loaded with the antibiotic

Now its time to score!

Now that the host cell has the recombinant DNA the “goal” is ready to be scored!

The purpose of the clones is to produce a “library” of specific gene sequences for researches to use

See slide 8

13.3 Cloned Libraries

Libraries are resources for gene studiesTo make a human genomic library using bacterial

plasmids ~8 million plasmid clones would be needed (plasmids generally can carry DNA fragments of about 1700 base pairs long)

YACs (yeast artificial chromosomes) are vectors that use eukaryotic yeasts as host cells; YACs can carry DNA fragments of 1 million base pairs. A human genome library could be carried in just over 3000 clones

Search through 8 million or search through 3000?

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

Could there be an easier way?

YES! A technique invented in 1986 called polymerase

chain reaction (PCR).

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

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

Automated DNA sequencing

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

Chapter 13 Assignment

Questions 1 – 18, 21 #6 I’ll give hint