Transcript Biotech tools

Biotechnology
http://library.thinkquest.org/28599/analogies.htm
Biotechnology -- definition
The use of living organisms, or substances
from living organisms, to develop
agricultural, medicinal, or environmental
product or process.
 Some examples?
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Biotech tools
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Recombinant DNA:
fragment of DNA
composed of
sequences originating
from at least two
different
sources(organisms)
http://www.cliffsnotes.com/study_guide/Recombinant-DNA-and-Biotechnology.topicArticleId-8524,articleId-8439.html
Restriction endonuclease animation
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http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
35::535::/sites/dl/free/0072437316/120078
/bio37.swf::Restriction Endonucleases
Biotech tools
Restriction endonucleases (restriction
enzymes): enzymes that are able to cleave
double stranded DNA into fragments at
specific sequences
 Each type of restriction enzyme recognizes a
characteristic sequence of nucleotides that is
known as its recognition site
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How Restriction Enzymes Work
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Most recognition sites are four to eight base pairs long and are
usually a complementary palindromic sequence
The restriction enzyme EcoRI binds to the following base-pair
sequence:
5´-GAATTC-3´
3´-CTTAAG-5´
EcoRI scans a DNA molecule and only stops when it is able to
bind to its recognition site.
Once bound, it disrupts, via a hydrolysis reaction, the
phosphodiester bond between the guanine and adenine
nucleotides on each strand.
Subsequently, the hydrogen bonds of complementary base pairs in
between the cuts are disrupted
The result is a cut within a DNA strand, producing two DNA
fragments where once there was only one.
Biotech tools
EcoRI produces sticky ends
 Both fragments have DNA nucleotides that are
now lacking their respective complementary
bases
 SmaI produces blunt ends
 The ends of the DNA molecule fragments are
fully base paired
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http://schoolworkhelper.net/2010/07/restriction-endonucleases-or-restriction-enzymes/
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The following sequence of DNA was digested with
the restriction endonuclease SmaI:
5´-TCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTA-3´
3´-AGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAAT-5´
SmaI recognizes the sequence CCCGGG and cuts
between the C and the G.
(a) Identify the location of the cuts.
(b) How many fragments will be produced if SmaI
digests this sequence?
(c) What type of ends does SmaI produce?
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Biotech tools: Methylases
Restriction endonucleases must be able to
distinguish between foreign DNA and the
genetic material of their own cells.
 Methylases are specific enzymes that, in
prokaryotes, modify the recognition site of a
restriction endonuclease by placing a methyl
group on one of the bases,
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https://netfiles.uiuc.edu/yxpan/www/Nutrigenegroup/Teaching.html
Methylation
Methylating a base prevents the restriction
endonuclease from cutting the DNA into
fragments.
 Methylases allow the molecular biologist to
protect a gene fragment from being cleaved
in an undesired location.
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Biotech tools: DNA Ligase
Two fragments of nucleic acids generated
using the same restriction enzyme, will
naturally be attracted to each other at
their complementary sticky ends
 Hydrogen bonds will form between the
complementary base pairs, but this is not a
stable arrangement.
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DNA ligase
The phosphodiester
linkage between the
backbones of the
double strands must be
reformed.
 DNA ligase is the
enzyme used for joining
the cut strands of DNA
together.
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http://www.biotechlearn.org.nz/themes/dna_lab/images/dna_ligation
Biotech tools: gel electrophoresis
Once a gene has been excised from its
source DNA, it must be separated from the
remaining unwanted fragments
 Gel electrophoresis takes advantage of the
chemical and physical properties of DNA
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DNA properties
DNA is negatively charged.
 The molar mass of each nucleotide pair is
relatively consistent.
 The only difference between two fragments
of DNA that are of differing lengths is the
number of nucleotides.
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Gel electrophoresis
DNA that has been
subjected to restriction
endonuclease digestion will
be cleaved into fragments
of different lengths.
 The shorter the fragment
is, the faster it will travel
because of its ability to
navigate through the pores
in the gel easily
 Larger fragments are
hampered by their size.
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http://www.web-books.com/MoBio/Free/Ch9C.htm
http://universe-review.ca/R11-16-DNAsequencing.htm
Gel electrophoresis
Gel electrophoresis takes advantage of DNA’s
negative charge.
 A solution containing different-size fragments to be
separated is mixed with a loading dye containing
glycerol and is placed in a well in the gel
 The gel itself is usually a square or rectangular slab
and consists of a buffer containing electrolytes and
agarose, or possibly polyacrylamide.
 Using direct current, a negative charge is placed at
one end of the gel where the wells are, and a positive
charge is placed at the opposite end of the gel.
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Gel electrophoresis
The negatively charged DNA
will migrate toward the
positively charged electrode
 The shorter fragments
migrating faster than the
longer fragments
 Small molecules found within
the loading dye migrate
ahead of all the DNA
fragments.
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http://www.docstoc.com/docs/21969430/AGAROSE-GEL-ELECTROPHORESIS-TO-SEPARATE-DNA-FRAGMENTS-After
Gel electrophoresis
Once gel electrophoresis is complete, the DNA
fragments are made visible by staining the gel
 The most commonly used stain is ethidium
bromide which is a molecule that fluoresces
under ultraviolet (UV) light and is able to insert
itself among the rungs of the ladder of DNA.
 When the gel is subjected to UV light, the bands
of DNA are visualized because the ethidium
bromide is inserted among the nucleotides.
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http://en.wikipedia.org/wiki/Gel_electrophoresis
Gel electrophoresis
The size of the fragments is then
determined using a molecular
marker as a standard.
 The molecular marker, which
contains fragments of known
size, is run under the same
conditions (in the same gel) as
the digested DNA.
 The resulting graph can be used
to determine the size of the
Digital image of 3 plasmid
restriction digests run on a 1% w/v
unknown fragments through
agarose gel, 3 volt/cm, stained with
ethidium bromide. The DNA size
interpolation.
marker is a commercial 1 kbp
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ladder.
Gel electrophoresis
a researcher is able to estimate the size
of a desired fragment
 the desired fragment can be excised out
of the gel
 The region containing the desired
fragment size can be purified for further
use
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Genetic engineering experiment
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http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
35::535::/sites/dl/free/0072437316/120078
/bio38.swf::Early Genetic Engineering
Experiment
Steps in cloning a gene
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http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
35::535::/sites/dl/free/0072437316/120078
/micro10.swf::Steps in Cloning a Gene
Biotech tools: plasmids
Plasmids are small, circular, double-stranded
DNA molecules that naturally exist in the
cytoplasm of many strains of bacteria.
 Bacteria are able to express foreign genes
inserted into plasmids
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http://lc-molecular.wikispaces.com/Isolating+Bli-1
Plasmids
The bacterial cell benefits from the
presence of plasmids.
 Plasmids often carry genes that confer:
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◦ antibiotic resistance
◦ resistance to toxic heavy metals, such as
mercury, lead, or cadmium
Using a plasmid
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Restriction endonucleases
are used to splice a foreign
gene into a plasmid.
Artificial plasmids have been
engineered to contain a
unique region that can be cut
by many restriction enzymes.
Recognition sites are present
only once in the plasmid
One cut results in the
circular plasmid becoming
linear.
Biotech tools: transformation
The introduction of DNA from another
source is known as transformation
 A bacterium that has taken in a foreign
plasmid is referred to as being transformed
 If a bacterium readily takes up foreign DNA,
it is described as a competent cell.
 Bacteria can be chemically induced in the
laboratory to become competent with the
aid of calcium chloride.
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Transformation
Bacterial cells are suspended in a solution of
calcium chloride at 0°C.
 Positively charged calcium ions stabilize the
negative charges of the phosphates on the
membrane
 The low temperature “freezes” the cell
membrane, making it more rigid.
 This stabilizes the cell membrane both
physically and chemically,
 Next the plasmid DNA is introduced into the
solution
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Transformation
The entire solution is subjected to a quick heat
shock treatment of 42°C that lasts for
approximately 90 seconds
 The outside environment of the cell is now at a
slightly higher temperature than the inside of
the cell.
 The resulting draft sweeps the plasmids into
the bacterial cell through pores in its
membrane.
 Finally, the bacterial cells are incubated in a
nutrient media suspension at a temperature of
37°C to recover
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Creating competent cells
Electroporators—chambers that subject the
bacteria to an electric shock which loosens
the structure of the cell walls and allows
foreign DNA to enter
 Modern electrical “gene guns” are used to
“shoot” DNA through the cell wall and
membrane of plant cells.
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Gene gun
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html
Electroporator
http://biology200.gsu.edu/core/Manuals/Electroporator.html
Biotech tools: selective plating
Selective plating
Selective plating is a method that can be
used to isolate the cells with recombinant
DNA
 If the transformation is a success, the
bacteria will be able to grow on media that
contain the antibiotic.
 If no growth is observed, the bacteria were
not transformed and were eliminated by the
antibiotic.
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Selective plating
It is necessary to check that the foreign gene
actually exists in the transformed bacteria.
 Colonies of bacteria are selected that have
been transformed.
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Selective plating
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Individual colonies allowed to proliferate in
liquid media until enough bacterial cells can be
harvested to extract a suitable amount of
plasmid DNA.
The extracted plasmid DNA is subjected to a
restriction enzyme digestion to release the
cloned fragment from the vector.
The DNA is run through electrophoresis to
determine if the expected pattern of bands is
observed on the gel
The Major Steps in the Cloning of DNA
1. Generation of DNA fragments using
restriction endonucleases
2. Construction of a recombinant DNA
molecule
• The target gene fragment is ligated to a DNA
vector
3. Introduction into a host cell
• Bacterial host cells can be manipulated to take up
the recombinant DNA using electroporators, gene
guns, or classical transformation protocols, such as
calcium chloride
The Major Steps in the Cloning of DNA
4. Selection
• Cells that have been successfully transformed with
the recombinant DNA must be isolated.
• The desired cells are usually chemically selected by
the presence of a marker (e.g. antibiotic resistance) on
the vector.
• Growth of colonies on media containing the chemical
indicates successful transformation of the recombinant
DNA vector.
• Individual colonies are isolated from media containing
the chemical and are grown in culture to produce
multiple copies (clones) of the incorporated
recombinant DNA.