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Gene Technology

Basic steps in DNA extraction

Lysis:
• Detergents
• Organic solvent
• Proteases
(lysozyme)
• Heat

“cell extract”

Genomic DNA prep: removing proteins and
RNA

chloroform

Need to mix gently! (to avoid shearing breakage of the
genomic DNA)
Add the enzyme RNase A to degrade RNA in the aqueous
layer

Precipitation of DNA
Precipitate out of DNA with isoproproanol

Agarose gel electrophoresis
• Agarose Gel Electrophoresis: separate and visualize DNA
fragments based on size
• Agarose is isolated from seaweed and when melted in a
buffer solution and poured into a horizontal tray and as it
cools it will form a semisolid gel containing small pores
through which DNA will travel
• The percentage of agarose used to make the gel
determines the ability of the gel to separate DNA
fragments of different sizes
• (gel % range from 0
...
5%) resolves larger size fragments

Agarose gel electrophoresis
• Agarose Gel Electrophoresis
– To run a gel, it is submerged in a buffer solution that
conducts electricity
– DNA is loaded into small depressions called wells at
the top of the gel
– Electric current is applied through electrodes at
opposite ends of the gel
• DNA migrates according to its charge and size
• Rate of migration through the gel depends on the size of the
DNA because the sugar phosphate backbone makes it
always negatively charged
• DNA migrates toward positive pole and is repelled by
negative pole

Agarose gel electrophoresis
• Agarose Gel Electrophoresis
• Migration distance is inversely proportional to size
of DNA fragment
– Large fragments migrate slowly; smaller fragments
migrate faster
– Tracking dye is added to the samples to monitor DNA
migration during electrophoresis
– DNA can be visualized after electrophoresis by the
addition of DNA staining dyes
• Ethidium bromide: intercalate between DNA base pairs and it
fluoresces under ultraviolet light
• Then a picture can be taken to document the gel results

Agarose gel electrophoresis

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...

Introduction to Recombinant
Technology and DNA Cloning

DNA

• Restriction Enzymes
Primarily found in bacteria (they use these for defense)
– Cut DNA by cleaving the phosphodiester bond that joins
adjacent nucleotides in a DNA strand
– There are 4 or 6 bp cutters because they recognize
restriction sites with a sequence of 4 or 6 nucleotides

Recognition site – specific base sequence on DNA where
a restriction enzyme binds
...

example: RACECAR or GAATTC
CTTAAG

• Each restriction enzyme has its own unique recognition site
...

Introduction to
Technology
and

Recombinant DNA
DNA
Cloning

• Restriction enzymes
a
...
Some cut DNA to generate fragments with
double-stranded ends called "blunt" ends

1
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Introduction to Recombinant DNA
Technology and DNA Cloning
• Plasmid DNA – small circular pieces of DNA
found primarily in bacteria
• Are considered extrachromosomal DNA because
they are in the cytoplasm in addition to the
bacteria chromosome
• Are small approximately 1 to 4 kb
• Can replicate independently of chromosome
• Can be used as vectors – pieces of DNA that can
accept, carry, and replicate other pieces of DNA

1
...
Introduction to Recombinant DNA
Technology and DNA Cloning
• Transformation of Bacterial Cells
– very inefficient process
– A process for inserting foreign DNA into bacteria
•
•
•
•

Treat bacterial cells with calcium chloride
Add plasmid DNA to cells chilled on ice
Heat the cell and DNA mixture
Plasmid DNA enters bacterial cells and is replicated and express
their genes

– electroporation
– Apply brief pulse of high voltage electricity to create tiny
holes in the bacteria cell wall that allow the DNA to enter

1
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Antibiotic selection – plate transformed cells on plates
containing different antibiotics to identify recombinant
bacteria and non-transformed bacteria
– Does not select for plasmid containing foreign DNA vs
...
Introduction to Recombinant DNA
Technology and DNA Cloning
• Selection
of
transformation

recombinant

bacteria

after

2
...
Introduction to Recombinant DNA
Technology and DNA Cloning

Producing Clones of Cells Carrying
Recombinant Plasmids
Several steps are required to clone the hummingbird
β-globin gene in a bacterial plasmid
-The hummingbird genomic DNA and a bacterial
plasmid are isolated
-Both are cut with the same restriction enzyme
-The fragments are mixed, and DNA ligase is added
to bond the fragment sticky ends

-Some recombinant plasmids now contain
hummingbird DNA
-The DNA mixture is added to bacteria that have
been genetically engineered to accept it
-The bacteria are plated on a type of agar that
selects for the bacteria with recombinant
plasmids
-This results in the cloning of many hummingbird
DNA fragments, including the β-globin gene

Hummingbird cell
ampR

Bacterial plasmid
gene

lacZ gene
Restriction
site
Sticky
ends

Gene of
interest
Hummingbird DNA
fragments

Recombinant plasmids Nonrecombinant
plasmid

Bacteria carrying
plasmids

RESULTS

Colony carrying nonrecombinant plasmid
with intact lacZ gene

Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones

1
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Appropriate restriction sites
2
...
Run on gel to separate fragments

4
...
Ligate with cut vector
6
...
Selection
...
Grow up colonies
...
Isolate plasmid DNA
...
Cut with RE to confirm presence of foreign DNA
...
Run on gel to identify recombinant plasmids
...
Run gel
Vector alone (no insert) - 1 band
4 kb
Vector + insert - 2 bands
4 kb AND 2 kb

2
...
What Makes a Good Vector?

3
...

• Paired set of Forward and Reverse Primers are added – short
single-stranded DNA oligonucleotides (20–30bp long)
– Primers are complementary to nucleotides flanking opposite ends of
target DNA

• Reaction tube is placed in an instrument called a thermocycler

3
...
Denaturation – heat to 94 °C to 96 °C
2
...
Extension (elongation) – DNA Pol copies target DNA at
70 to 75 °C
– At the end of one cycle, the amount of DNA has
doubled
– Cycles are repeated 20–30 times

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How Do You Identify and Clone a Gene of
Interest?
• Advantage of PCR:
• Can amplify millions of copies of target DNA from
small amount of starting material in short period of
time
• To calculate the number of copies of target DNA
starting with 1 molecule of DNA use this equation
2N in which N represents number of PCR cycles
• Assume you want to do 22 PCR cycles to
amplify your DNA insert, how many copies of
DNA will you have at the end of your PCR?

3
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How Do You Identify and Clone a Gene of
Interest?
• Cloning PCR Products
– Is rapid and effective

– Disadvantage
• Need to know something about the DNA sequence that
flanks the gene of interest to design primers

3
...
Applications
Technology

of

Recombinant

DNA



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