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Chapter 3

Recombinant DNA
Technology and Genomics
Agarose Gel Electrophoresis

 Electrophoresis is a molecular technique that separates


nucleic acids and proteins based on:

Size
and

+-+Charge+-+
Agarose Gel Electrophoresis

DNA is a negatively charged molecule and


therefore is attracted to positive charges.
Agarose Gel Electrophoresis
Agarose provides a matrix through which DNA
molecules migrate.
 Larger molecules move through the matrix slower
than small molecules
 The higher the concentration of agarose, the better
the separation of smaller molecules
Agarose Gel Electrophoresis
How to make an agarose gel:
1. Weigh out a specified amount of agarose powder.
2. Add the correct amount of buffer.
3. Dissolve the agarose by boiling the solution.
4. Pour the gel in a casting tray.
5. Wait for the gel to polymerize.
Agarose Gel Electrophoresis
How to make an agarose gel:
6. Place gel in chamber and cover with buffer
7. Add loading dye to the sample
8. Load sample on to the gel.
Agarose Gel Electrophoresis
How to make an agarose gel:
9. Stain the gel
10. Take a picture of the gel
11. Analyze results
Agarose Gel Electrophoresis

Electrophoresis Animation
Recombinant DNA

Recombinant DNA technology


 Allows DNA to be combined from different sources
 Also called genetic engineering or transgenics
Recombinant DNA
 Vector – DNA source which can replicate and is used to
carry foreign genes or DNA fragments.

 Recombinant DNA – A vector that has taken up a


foreign piece of DNA.
Restriction Enzymes

 Restriction enzyme – an enzyme which binds to DNA at


a specific base sequence and then cuts the DNA
 Restriction enzymes are named after the bacteria from
which they were isolated.

• Bacteria use restriction enzymes to chop up foreign viral DNA


Restriction Enzymes
 Recognition site – specific base sequence on DNA where
a restriction enzyme binds.
• All recognition sites are palindromes, which means they read
the same way forward and backward.
example: RACECAR or GAATTC
CTTAAG
• Each restriction enzyme has its own unique recognition site.
Restriction Enzymes
Restriction Enzymes
 After cutting DNA with restriction enzymes, the
fragments can be separated on an agarose gel.
• The smaller fragments will migrate further than the longer
fragments in an electric field.
• The bands are compared to standard DNA of known sizes. This
is often called a DNA marker, or a DNA ladder.
Restriction Enzymes

Running a Restriction digest on an agarose gel


Restriction Enzymes
After analyzing your results, you draw a
restriction map of the cut sites.
• A restriction map is a diagram of DNA showing the
cut sites of a series of restriction enzymes.
Restriction Enzymes
Restriction Enzymes
Restriction Enzymes

Most restriction enzymes cut within the


recognition site.
When restriction enzymes cut in a zig zag
pattern, sticky ends are generated.
Restriction Enzymes
 Overhanging sticky ends will
complementarily base pair,
creating a recombinant DNA
molecule.

 DNA ligase will seal the nick


in the phosphodiester
backbone.
Restriction Enzymes

Restriction Enzyme Animation


Transformation

Transformation – the process by which


organisms take up and express foreign DNA

Griffith’s experiment
Transformation
Bacterial Transformation
 Bacteria, such as E.coli, can take up and express
foreign DNA, usually in the form of a plasmid.
Transformation

Gene cloning – using bacterial transformation to


make lots of copies of a desired gene.

Gene Cloning Animation


Transformation
Steps of Bacterial Transformation
1. Choose a bacterial host
a. E. coli is a model organism
i. Well studied
ii. No nuclear membranes
iii. Has enzymes necessary for replication
iv. Grows rapidly (20 min. generation time)
v. Inexpensive
vi. Normally not pathogenic
vii. Easy to work with and transform
Transformation
Steps of Bacterial Transformation
2. Choose a plasmid to transform
a. Characteristics of a useful plasmid
i. Single recognition site
• Plasmid only cuts in one place, so this ensures that the plasmid is
reformed in the correct order.
ii. Origin of replication
• Allows plasmid to replicate and make copies for new cells.
iii. Marker genes
• Identifies cells that have been transformed.
 gene for antibiotic resistance – bacteria is plated on media
with an antibiotic, and only bacteria that have taken up a
plasmid will grow
 gene that expresses color – bacteria that have taken up a
recombinant plasmid are a different color than bacteria
that have taken up a NONrecombinant vector.
Transformation
Steps of Bacterial Transformation
3. Prepare bacterial cells for transformation
a. Treat with calcium chloride – softens the phospholipid
bilayer of the cell membrane, which allows the plasmid to
pass through
b. Electroporation – brief electric pulse
c. Directly inject plasmid into bacterial host
Transformation
Steps of Bacterial Transformation
4. Plate transformation on
appropriate media
a. Contains nutrients for bacteria and
antibiotic to distinguish transformed
bacteria from NONtransformed
bacteria
5. Incubate plates overnight
a. E.coli grows at body temp. (37 °C)
6. Analyze plates

Gene Cloning Animation


Gene Cloning
What makes a good vector?
Gene Cloning
What makes a good vector?
Gene Cloning
How do you identify and clone a gene of interest?
• BUILD A LIBRARY!!
• DNA library – a collection of cloned DNA fragments from a
particular organism
• Can be saved for a relatively long period of time and screened to
pick out different genes of interest
• Two types of libraries
1. Genomic library – contains DNA sequences from entire genome
2. cDNA library – contains DNA copies of mRNA molecules expressed

Construction of a DNA library Animation


Gene Cloning
Gene Cloning
Steps to screen a library
1. Plate cells and transfer to nylon
membrane
2. Lyse bacterial cells
3. Denature DNA
4. Add radioactively labeled probe
that is complementary to gene of
interest
Gene Cloning
Steps to screen a library
5. Wash off non-specifically bound
probe
6. Expose membrane to x-ray film
7. Align exposed film with original
plate
8. Grow cells containing gene of
interest in culture.
Gene Cloning

Rarely is an entire gene cloned in one piece,


even in a cDNA library, therefore must “walk”
the chromosome until a start and stop codon
are found.
Sequencing

Sequencing – determining the


order and arrangement of G’s,
A’s, T’s and C’s in a segment of
DNA.
Sequencing

Let’s review replication…..


Sequencing

The Sanger sequencing method uses dideoxy-


nucleotides to generate all possible fragments of
the DNA molecule to be sequenced.

deoxynucleotide dideoxynucleotide
Sequencing

Set up four different reactions:


Sequencing

Load the four reactions in different wells of a


polyacrylamide gel to separate the fragments
Sequencing

Sequencing Animation
Human Genome Project

Initiated in 1990 with plan to


identify all human genes
• Analyze genetic variation
among humans
• Map and sequence genomes
of model organisms
• Develop new lab technology
• Disseminate genome
information
• Consider ethical, legal, and
social issues that accompany
genetic research
Human Genome Project

Francis Collins Craig Venter


Human Genome Project

Consider ethical, legal and social issues


• Who owns your DNA?
Human Genome Project
Develop new lab technology
• Automated Sequencing
Human Genome Project

Disseminate genome information


• GenBank database
Human Genome Project
Analyze genetic variation among humans
• The genome is approximately 99.9% identical
between individuals of all nationalities and
backgrounds.
Human Genome Project
 Map and sequence genomes of model organisms
• E.coli

• Arabidopsis thaliana

• Saccharomyces cerevisiae

• Drosophila melangaster

• Caenorhabditis elegans

• mus musculus
PCR

Polymerase chain reaction (PCR)


 A lab technique used to amplify segments of DNA

"PCR has transformed molecular biology


through vastly extending the capacity to
identify, manipulate and reproduce DNA. It
makes abundant what was once scarce -- the
genetic material required for
experimentations."
PCR
 Reaction requirements
 Template DNA – total genomic
DNA isolated from an organism
that contains a target region to be
amplified
 DNA primers - Short pieces of
single stranded DNA that flank the
target
 Taq DNA polymerase - Attaches
nucleotides on the growing strand
of DNA
 Nucleotides (GATC) – Polymerase
adds complementary nucleotides
to the template
PCR

Reactions are placed in a machine called a


thermal cycler. The machine cycles through
three temperatures.
PCR

1. Heat samples to 94°C for a minute or so to


denature the double stranded template DNA.
PCR

2. Drop temperature to around 50 or 60°C to allow


primers to anneal.
PCR

3. Maintain temperature at 72°C for a minute or two to


allow the polymerase to elongate the new DNA strands.
PCR

The thermal cycler repeats the denaturing,


annealing, and elongating temperatures
approximately 30 times.

PCR Animation
PCR

PCR amplification is logarithmic, meaning the


number of copies of the target is doubled every
cycle. (2n)
PCR

PCR animation
PCR
Cloning by PCR
• Design primer specific for gene of interest (must
know some of the sequence)
• Can use a T-vector because Taq polymerase adds an
A to the 3’ end of sequence
Applications of Recombinant DNA Technology
Chromosomal Location and Gene
Copy Number

Fluorescence in situ hybridization (FISH)


Chromosomal Location and Gene
Copy Number
Southern Blot - molecular technique where DNA is
transferred onto a membrane from an agarose
gel and a probe is hybridized.
Southern Blot

The first step in preparing a Southern Blot is to


cut genomic DNA and run on an agarose gel.
Southern Blot

The next step is to blot or transfer single stranded


DNA fragments on to a nylon membrane.
Southern Blot
 The next step is to hybridize a radioactively labeled DNA
probe to specific sequences on the membrane.
Southern Blot

The last step is to expose the radioactively


labeled membrane to a large sheet of film.
You will only visualize bands where the probe
hybridized to the DNA..
Southern Blot

Southern Blot Animation


Studying Gene Expression
 Northern Blot
• Isolate RNA from tissue of interest
• Separate on agarose gel
• Blot onto nylon membrane
• Hybridize probe specific for desired
transcript
• Expose on film
 Reverse Transcription PCR (RT-PCR)
• Used if RNA produced is below detection
level for Northern blot
• Isolate RNA from tissue of interest
• Convert into double stranded cDNA
• Amplify by PCR
• Run on agarose gel
Studying Gene Expression

Real Time PCR (qPCR)


• Eliminates the need for
running agarose gels
• Is quantitative
Studying Gene Expression

Gene microarray

Microarray animation