Download DNA Sequencing

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 sites
Restriction 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 studies
To 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