Transcript DNA Fingerprinting and Gel Electrophoresis Notes June 2, 2016

DNA Fingerprinting and Gel
Electrophoresis Notes
June 2, 2016
Transgenics
• Genetic engineering
makes it possible to
transfer DNA sequences
from one organism to
another
• REMEMBER- DNA is
universal
• A gene taken from one
species can be replicated
in another species.
Recombinant DNA
• Recombinant DNA contains DNA
from 2 or more different sources.
• So how does DNA get from one species
to another?
• Vector: something used to transfer
DNA from one organism to another
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Microinjection
Gene Gun
Virus
Bacteria Plasmid
Bacteria as vectors
• Bacteria are used because they have plasmids
• A plasmid is a circular
piece of DNA that exists
apart from the chromosome
and replicates independently
of it.
DNA fingerprints
• Made by cutting DNA in very specific places.
• How do we cut DNA?
Do you remember
• Restriction Enzymes
what a restriction
enzyme is?
What are Restriction Enzymes?
• Proteins
• In nature: Used by bacteria to cut viral
DNA
• They “restrict” the growth of viruses
• http://www.dnalc.org/resources/anima
tions/restriction.html
Sticky Ends
• Most restriction enzymes cut
DNA with a staggered cut
• The staggered cuts leave the
DNA with end pieces “sticking
off”
▫ We call these “sticky ends”
▫ These exposed N-bases will want
to join with other complimentary
exposed bases
So what ?
• What do you predict could happen if two pieces
of DNA are cut with the same restriction
enzyme???
▫ YES! They will have the same “sticky ends”
▫ How could we use this???
• Restriction Enzymes can be used to make
Recombinant DNA.
• The gene of interest can be isolated using
Restriction Enzymes
Restriction Enzymes can be used to make
RECOMBINANT DNA!
• The gene you are interested in inserting (aka the
“gene of interest”) can be cut using a restriction
enzyme.
• What will happen if I also cut the other
organisms DNA with the same Restriciton
Enzyme?
Making Recombinant DNA…
• Once the gene is isolated, how do we join it with
the organism’s DNA?
• Cut the organism’s DNA with the same
restriction enzyme…why
▫ The sticky ends will naturally be attracted to each
other
• Add DNA LIGASE: an enzyme that seals the
fragments together
Restriction Enzymes
• Cut strands of DNA at specific nucleotide
sequences
• There are many different restriction enzymes
that each cut DNA at different nucleotide
sequences
• Usually occurs at a palindrome
5’ GAATTC
3’ CTTAAG
What is a palindrome in English?
• Palindromes can be read backwards and
forwards the same way.
• Give me an example of a name that is a
palindrome:
▫ Hannah
• It’s a little more tricky in DNA because the
strand itself isn’t what must be backwards and
forwards…
DNA palindromes
• A DNA palindrome reads exactly opposite its
complementary strand
• Ex- CAGAC is not a palindrome but CCCGGG
is. Why?
• Let’s look at the complementary strand:
• CAGAC
CCCGGG
• GUCUG
GGGCCC
So back to DNA fingerprinting…
I. DNA Fingerprinting
A. DNA is cut using ____________________.
1. Because no two individuals have identical
DNA, no two individuals will have the same
length fragments
B. Once the restriction enzyme has successful cut
the DNA into fragments, the next step in
analyzing the DNA is to use a gel
electrophoresis
II. Gel Electrophoresis
A. Gel electrophoresis is a technique used to
separate the DNA fragments according to their
size
B. DNA fragments are placed in wells on a sheet
of agarose gel
1. Gel is slightly porous and allows fragments to
move through the gel at a rate relative to their
size
C. An electric current is applied to the sheet
D. DNA is negatively charged and will move in an
electric field toward the positive pole
▫
Which size fragments do you expect to move
faster through the gel?
1. Smaller fragments move through the gel at a
faster rate compared to the larger pieces
E. Bands will be produced on the gelatin where the
fragments accumulate
1. The more fragments that accumulate in one spot,
the thicker the line will be in the gel
F. DNA bands must be stained to make them visible
III. Uses for DNA Fingerprinting and Gel
Electrophoresis
A. Used in forensics as a
way to match a sample
from a crime scene to a
suspect’s DNA
B. Used in paternity cases
where the father is
unknown
1. Each fragment in the
child’s DNA must
match at least one of
the parent’s
fragments
C. Used to analyze genes to test for presence of
different alleles
1. If a person was homozygous for a trait, the DNA
would be cut by the same restriction enzyme in
the same place because alleles are the same
a. Fragments would be at the same point when run
through a gel electrophoresis
Allele from Mom
Allele from Dad
2. If a person was heterozygous for a trait, the DNA
would be cut by the same restriction enzyme in
different places because the alleles are different
a. Fragments would not be at the same point in the
gel electrophoresis
Allele from Mom
Allele from Dad