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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!