Power Point - Restriction Enzymes

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 digests (cuts) DNA at a specific sequence = restriction site

Palindrone

• 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 CTTAAG

GAATTC CTTAAG

1 Digestion G CTTAA

AATTC G

2 Annealing of sticky ends

Ligase

GAATTC CTTAAG

3 Ligation

4 Recombinant DNA GAATTC CTTAAG

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Agarose Electrophoresis Loading • Electrical current carries negativelycharged 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

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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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