[ELAINE] RESTRICTION ENZYMES AND MAPPING

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27 Terms

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Restriction Enzymes

DNA-cutting enzymes which recognizes one or a few target sequences

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Endonucleases

break the sugar phosphate backbone at internal sites

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Restriction Enzymes

endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone

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Genetic Engineering

stimulated by the discovery of deoxyriboendonucleases or endonucleases

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BamHI

isolated from Bacillus amyloliquefaciens H

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HindIII

isolated from Haemophilus influenzae Rd

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SmaI

isolated from Serratia marcescens

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DNA recombination

combination of two genes in a single host

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DNA Cloning

set of procedures that uses living cells to make many identical copies of a DNA fragment

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Plasmid

circular form of dsDNA which is a good template to introduce genes

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pBR322

antimicrobial resistance gene

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EcoR1

restriction enzyme that cuts pBR322

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Type I Restriction Enzymes

have both nuclease and methylase activity in a single enzyme. Bind to host-specific DNA that contains methylated adenines

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Type II Restriction Enzymes

used most frequently in the laboratory; do not have inherent methylation activity in the same molecule as the nuclease activity

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Type II Restriction Enzymes

palindromic/bilateral symmetry

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Prokaryotes

Type II Restriction Enzymes have been found in almost all

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Overhangs

cuts the DNA duplex with a staggered separation at the recognition site; produces sticky ends

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Blunt

cuts the DNA duplex at the same place on both strands

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Type III Restriction Enzymes

resemble type I enzymes in their ability to methylate and restrict (cut) DNA; do not always able to provide complete restriction enzyme digestion

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Type III Restriction Enzymes

adenine methylation occurs on only one strand.

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Type IV Restriction Enzymes

can only cleave methylated DNA and sequence specificity is weak

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Type V Restriction Enzymes

Requires guide DNA to target specific sequences and it is these that are being modified or used in genome engineering methods such as TALENS or CRISPR-Cas9

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CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

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restriction endonucleases

provide a convenient tool for molecular characterization of DNA

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Restriction map

DNA is exposed to several restriction enzymes separately and then in particular combinations

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pattern of fragments

produced by restriction enzyme digestion that can be used to identify that DNA and monitor changes in size,structure or DNA sequence

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Restriction Fragment Length Polymorphisms (RFLPs)

resulting differences in the size or number of restriction fragments; used for the clinical analysis of structural changes in chromosomes associated with disease