Download Ch. 13 Bioengineering

Bioengineering
Biology
Ch.13
Ms. Haut
The Tools of Molecular Biology
• Scientists use their knowledge of the
structure of DNA and its chemical
properties to study and change DNA
molecules.
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The Tools of Molecular Biology
• Scientists use different techniques to:
– extract DNA from cells
– cut DNA into smaller pieces
– identify the sequence of bases in a DNA
molecule
– make unlimited copies of DNA
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The Tools of Molecular Biology
• In genetic engineering, biologists make
changes in the DNA code of a living
organism.
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The Tools of Molecular Biology
• DNA Extraction
– DNA can be extracted from most cells by a
simple chemical procedure.
– The cells are opened and the DNA is
separated from the other cell parts.
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The Tools of Molecular Biology
• Cutting DNA
– Most DNA molecules are too large to be
analyzed, so biologists cut them into smaller
fragments using restriction enzymes.
• Enzymes found in bacteria used to destroy phage
DNA
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The Tools of Molecular Biology
• Each restriction enzyme cuts DNA at a specific
sequence of nucleotides.
The Tools of Molecular Biology
• A restriction enzyme will cut a DNA
sequence only if it matches the sequence
precisely.
The Tools of Molecular Biology
• Separating DNA
– In gel electrophoresis, DNA fragments are placed at one
end of a porous gel, and an electric voltage is applied to
the gel.
– When the power is turned on, the negatively-charged DNA
molecules move toward the positive end of the gel.
BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece,
Mitchell, and Taylor, ©2003.
The Tools of Molecular Biology
• Gel electrophoresis can be used to
compare the genomes of different
organisms or different individuals.
• It can also be used to locate and identify
one particular gene in an individual's
genome.
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The Tools of Molecular Biology
• DNA Fingerprinting
Dr. Alec Jeffreys
– A method of
developing a
person’s DNA
“profile,” similar to a
fingerprint.
– Pioneered in England
in 1984 by Dr. Alec
Jeffreys
First Forensic Use
• First used by law
enforcement in
England in the mid1980’s.
• DNA evidence
exonerated one man,
and convicted
another.
• Described in The
Blooding, by Joseph
Wambaugh
How does it work?
• 99.9% of your DNA is the same as
everyone else’s.
• The 0.1% that differs are a combination of:
– Gene differences (Differences in the genes
themselves)
– Differences in “polymorphic regions” between
the genes on the DNA.
How does it work?
• Certain points between the genes on the
DNA have repeating base sequences.
– For example:
ATTACGCGCGCGCGCGCGCTAGC
– These are called variable number tandem
repeats (VNTRs for short)
How does it work?
• Everyone has VNTRs at the same place in
their DNA, but they are different lengths
for different people.
– For example:
Person 1: ATTACGCGCGCGCGCGCGTAGC
(7 repeats)
Person 2: ATTACGCGCGCGCGTAGC
(5 repeats)
To Make a DNA Fingerprint…
• First, we use restriction enzymes to
chop the DNA up into millions of
fragments of various lengths.
– Some of the fragments contain STRs;
some do not. The ones that do are different
lengths for different people.
To Make a DNA Fingerprint…
BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by
Campbell, Reece, Mitchell, and Taylor, ©2003.
• There are slight differences in DNA
sequences as seen in individuals of the
same species
To Make a DNA Fingerprint…
• Next, we use gel
electrophoresis to sort
the DNA fragments by
size.
Gel Electrophoresis
• Method for sorting
proteins or nucleic
acids on the basis
of their electric
charge and size
Gel Electrophoresis
Electrical current
carries negativelycharged DNA through
gel towards positive
electrode
• Agarose gel sieves
DNA fragments
according to size
•
–
Small fragments move
farther than large
fragments
BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece,
Mitchell, and Taylor, ©2003.
Gel Electrophoresis
BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece,
Mitchell, and Taylor, ©2003.
1
To Make a DNA
Fingerprint…
Restriction fragment
preparation
2
Gel electrophoresis
3
Blotting
4
Radioactive probe
Filter paper
Probe
5
Detection of radioactivity
(autoradiography)
Film
Figure 12.11C
BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece,
Mitchell, and Taylor, ©2003.
Restriction
fragments
• Finally, a radioactive
probe attaches to our
VNTRs. Only the
fragments with our
VNTRs will show up
on the gel.
To Make a DNA Fingerprint…
• Since VNTRS are
different lengths in
different people, this
creates a DNA
Fingerprint.
Two uses for DNA
Fingerprints...
• Forensics
DNA taken from crime
scenes (blood, semen, hair,
etc.) can be compared to
the DNA of suspects.
Real-life CSI!
Two uses for DNA
Fingerprints...
• Forensics
This is an example of a
gel that might be used to
convict a rape suspect.
Compare the “Sperm
DNA” to the “Suspect
DNA.” Which suspect
committed the rape?
Two uses for DNA
Fingerprints...
• Paternity Testing
Since all of our DNA
markers came from
either mommy or
daddy, we can use
DNA fingerprints to
determine whether a
child and alleged
father are
related…just like on
Maury Povich!
Two uses for DNA
Fingerprints...
• Look at the two “Child” markers
on this gel. Can they both be
matched up to either the
mother or the “alleged father?”
• Yes. This is a “positive” test for
paternity.
Two uses for DNA
Fingerprints...
• How about this gel? Do both
of the child’s markers match
either the mother or the
“alleged father.”
• No! The “alleged father” is not
this child’s biological parent.
Interpreting DNA Fingerprints
• Which child is not
related to the
mother?
• Son 2
• Which children
are not related to
the father?
• Daughter 2 and
Son 2
Interpreting DNA Fingerprints
• A blood stain was found
at a murder scene. The
blood belongs to which of
the seven possible
suspects?
Suspect 3
Interpreting DNA Fingerprints
• Who committed this
sexual assault?
Suspect 1
Interpreting DNA Fingerprints
• These DNA fingerprints
are from a mother, a
child, and two possible
biological fathers. Which
one is the daddy?
2nd alleged father
Interpreting DNA Fingerprints
• Mother, father, and
four children. Which
child is from a
different father?
Child 2
• Cutting and Pasting
– Short sequences of
DNA can be
assembled using DNA
synthesizers.
– “Synthetic” sequences
can be joined to
“natural” sequences
using enzymes that
splice DNA together.
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BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece,
Mitchell, and Taylor, ©2003.
Using the DNA Sequence
Using the DNA Sequence
Copyright Pearson Prentice Hall
BIOLOGY: CONCEPTS AND CONNECTIONS 4th
Edition, by Campbell, Reece, Mitchell, and Taylor,
©2003.
• These enzymes also make it possible to take a
gene from one organism and attach it to the
DNA of another organism.
• Such DNA molecules are sometimes called
recombinant DNA.
Using the DNA Sequence
• Making Copies
– Polymerase chain reaction (PCR) is a
technique that allows biologists to make
copies of genes.
– A biologist adds short pieces of DNA that are
complementary to portions of the sequence.
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Using the DNA Sequence
5
• DNA is heated to
separate its two
strands, then cooled
to allow the primers to
bind to singlestranded DNA.
• DNA polymerase
starts making copies
of the region between
the primers.
3
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
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3
5
5
3
3
5
Primers
New
nucleotides
Restriction enzymes are used to
a)
b)
c)
d)
extract DNA.
cut DNA.
separate DNA.
replicate DNA.
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During gel electrophoresis, the smaller the DNA
fragment is, the
a)
b)
c)
d)
more slowly it moves.
heavier it is.
more quickly it moves.
darker it stains.
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The DNA polymerase enzyme Kary Mullis found
in bacteria living in the hot springs of
Yellowstone National Park illustrates
a)
b)
c)
d)
genetic engineering.
the importance of biodiversity to biotechnology.
the polymerase chain reaction.
selective breeding.
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A particular restriction enzyme is used to
a)
b)
c)
d)
cut up DNA in random locations.
cut DNA at a specific nucleotide sequence.
extract DNA from cells.
separate negatively charged DNA molecules.
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During gel electrophoresis, DNA fragments
become separated because
a)
b)
c)
d)
multiple copies of DNA are made.
recombinant DNA is formed.
DNA molecules are negatively charged.
smaller DNA molecules move faster than larger
fragments.
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6
Why would someone use PCR?
a)
b)
c)
d)
To cut the DNA into fragments
To create recombinant
To make lots of copies of a DNA fragment
To make a transgenic organism
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