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Enzymes are the Tools of

DNA Technology

These enzymes are the Restriction Endonucleases

Restriction - Because for the way they work, they restrict virus
to only one host bacterial strain. They are also restricted to
acting on only specific DNA sequences

Endonuclease - They cut nucleic acids in the middle not just
the ends

Restriction Endonucleases
There are a number of different sub classes of
restriction endonucleases
Type I - Recognize specific sequences and cut DNA at a
nonspecific site > than 1,000 bp away
Type II - Recognize palindromic sequences and cut within
the palindrome at the sugar phosphate backbone
Type III - Recognize specific 5-7 bp sequences and cut
24-27 bp down stream of the site.

Type II are the most useful

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What is a Palindrome?
A palindrome is anything that reads the same forwards and
backwards:
English palindromes:
Mom
Dad
Tarzan raised Desi Arnaz rat.
Able was I ere I saw Elba (supposedly said by Napoleon)
Doc note I dissent, a fast never prevents a fatness, I diet on
cod.

DNA Palindromes
Because DNA is double stranded and the strands
run antiparallel, palindromes are defined as any
double stranded DNA in which reading 5’ to 3’
both are the same
Some examples:
The EcoRI cutting site:
5'-GAATTC-3'
3'-CTTAAG-5'
The HindIII cutting site:
5'-AAGCTT-3'
3'-TTCGAA-5'

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Enzyme Site Recognition
Restriction site

• Each enzyme Palindrone
digests (cuts)
DNA at a
specific
sequence =
restriction site
• Enzymes
recognize 4- or
6- base pair,
palindromic
sequences
(eg GAATTC)
Fragment 1 Fragment 2

5 vs 3 Prime Overhang
Enzyme cuts

• Generates 5 prime
overhang

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Gel Electrophoresis
Separates DNA (or RNA or Protein) fragments on the basis
of charge and size

The larger the fragment, the more difficulty it has moving
through gels

By placing DNA in a gel, then applying a voltage accross
the gel, the negatively charged DNA will move toward
the positive pole

Large fragments lag behind while small fragments move
throght the gel relatively rapidly

R. E.s and DNA Ligase
Can be used to make recombinant DNA
EcoRI
EcoRI GAATTC
GAATTC
CTTAAG CTTAAG

1 Digestion AATTC
G
G
CTTAA 2 Annealing of sticky ends
Ligase
GAATTC
CTTAAG
4 Recombinant DNA
3 Ligation GAATTC
CTTAAG

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Agarose
Electrophoresis
Loading
• Electrical
current carries
negatively-
charged DNA
through gel
towards
positive (red)
electrode
Buffer

Dyes

Agarose gel

Power Supply

Gel Electrophoresis

Wells

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Gel Electrophoresis
-
Wells
Large

Direction
of
DNA
Travel

Small

+

Analysis of Stained Gel

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DNA
Fingerprinting

Uses of Restriction
Endonucleases

Because restriction endonucleases cut specific
sequences they can be used to make “DNA
fingerprints” of different samples of DNA.

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Karyotyping
Indentifies
Genetic
Anomalies

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DNA Extraction:
DNA can be extracted from
almost any human tissue.
•Buccal cells from inside cheek for
paternity tests.
•Sources of DNA at crime scene: blood,
semen, hair follicle, saliva.
•DNA extracted from evidence is
compared to DNA from known individuals

An EcoR1 restriction enzyme

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RFLP Analysis:
•RF stands for Restriction Fragments. Those
are the fragments that were cut by restriction
enzymes.
•L stands for Length, and refers to the length
of the restriction fragment.
•P stands for Polymorphisms, a Greek term
for “many shapes”. The lengths of some of
the restriction fragments differ greatly
between individuals.
RFLP = Restriction Fragment Length
Polymorphism

Electrophoresis of these RFLP’s produce
different patterns of DNA bands.
With 3 billion base pairs in the human
genome, however, RFLP analysis would
produce a ‘smear’ of many similar sized
fragments.
Molecular biologists have identified regions
of the human genome where restriction
fragment lengths are highly variable
between individuals.

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VNTR alleles are highly variable regions of
human DNA.
•VNTR stands for ‘variable number of
tandem repeats.
•A tandem repeat is a short sequence
of DNA that is repeated at a specific
chromosomal locus.
•Tandem repeats are interspersed
throughout the human genome.

VNTR’s continued:
•The number of repeats at a given
place on a certain chromosome is
highly variable from one person to
another.
•The number of such repeats is
usually different on the paternal and
maternal members of the same
person’s chromosome pair.

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Red boxes represent the repeat unit and the blue lollipops
represent cut sites for a restriction endonuclease. (Here 3
different variants, may be 50 in reality).

The possible genotypes
are AA, BB, CC, AB,
BC, and AC

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•The RFLP markers most commonly used for DNA
profile analysis are found on chromosomes 1, 2, 4,
5, 10 and 17.

•These RFLP markers are named after their
locations on these chromosomes.

•For example, the marker on chromosome 2 is
called D2S44 (section 44 of chromosome 2).

•These chromosomal locations are also referred to
as DNA loci.

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•The Federal Bureau of Investigation (FBI) has
been a leader in developing DNA typing
technology for use in the identification of
perpetrators of violent crime.

•In 1997, the FBI announced the selection of 13
STR (short tandem repeat) loci to constitute the
core of the United States national database,
CODIS.

•All CODIS STRs are tetrameric repeat sequences.

•All forensic laboratories that use the CODIS
system can contribute to a national database.

How common or rare would this 13 locus DNA
profile be in the reference population?

In most cases, a "product rule" calculation can be
done by multiplying each individual probability
together

By combining the frequency information for all
13 CODIS loci, this frequency of this profile
would be 1 in 7.7 quadrillion Caucasians…that’s
1 in 7.7 x 1015 power!

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