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Enzymes are the Tools of Enzymes are the Tools of
DNA TechnologyDNA 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 Restriction EndonucleasesEndonucleasesThere 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?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 PalindromesDNA PalindromesBecause 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 RecognitionEnzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromicsequences (eg GAATTC)
Palindrone
Restriction site
Fragment 1 Fragment 2
5 vs 3 Prime Overhang5 vs 3 Prime Overhang
• Generates 5 prime overhang
Enzyme cuts
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Gel ElectrophoresisGel ElectrophoresisSeparates 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 accrossthe gel, the negatively charged DNA will move toward the positive pole
Large fragments lag behind while small fragments move throght the gel relatively rapidly
G
CTTAA
AATTC
G
1 Digestion
2 Annealing of sticky ends
3 Ligation
Ligase
G
CTTAA
AATTC
G
EcoRIEcoRI
R. E.s and DNA R. E.s and DNA LigaseLigaseCan be used to make recombinant DNACan be used to make recombinant DNA
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
4 Recombinant DNA
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AgaroseAgaroseElectrophoresisElectrophoresis
LoadingLoading•Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
Gel ElectrophoresisGel Electrophoresis
Wells
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Gel ElectrophoresisGel Electrophoresis
+
-
Direction
of
DNA
Travel
Wells
Small
Large
Analysis of Stained GelAnalysis of Stained Gel
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DNA
Fingerprinting
Uses of Restriction Uses of Restriction
EndonucleasesEndonucleases
Because restriction endonucleases cut specific
sequences they can be used to make “DNA
fingerprints” of different samples of DNA.
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KaryotypingKaryotyping
IndentifiesIndentifies
Genetic Genetic
AnomaliesAnomalies
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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
Molecular biologists have identified regions
of the human genome where restriction
fragment lengths are highly variable
between individuals.
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.
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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!