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Lecture 4

BIOL 5060 Lecture 4: 1:24_RecombinantDNATechnology_Hoffman

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Boston College
BIOL 5060

1/24_RecombinantDNATechnology_Hoffman 1/24/2017 3:02:00 PM Breaking DNA- restriction enzymes/ endonucleases • Exonucleases remove nucleotides from the end of the molecule • Type II- breaks at a specific location relative to a recognition sequence What’s the purpose of breaking DNA? 1. Diagnostic a. Identification/ confirmation of plasmids 2. To prepare for a DNA construction a. PCR reaction- to make a piece of DNA Today’s Topic…Working with Restriction Enzymes • Restriction enzymes are proteins- need to handle them with the idea that their biological activity shouldn’t be taken for granted o Do not vortex them!  denatures proteins o Do not dilute them into water!  Denatures proteins ▪ Can only dilute them into a buffered solution o Their activity is defined by units/ function of conditions of reactions—bugger, DNA concentration temperature o Partially purified agents ▪ Contaminants to worry about • Phosphatase- remove 5’ phosphate to prevent ligation • Other endonuclease activity o Too much DNA can prevent digestion ▪ A mock digest (everything except enzyme) can be useful control o Uncut plasma can produce 3 bonds ▪ Open circle (OC)= one strand of super-coiled was nicked/ broken (only one of the two strands/ not a complete break) all the super coils unravel ▪ Linear ▪ Super-coiled (SC) • What is the unit of a restriction enzyme? What does one unit represent in terms of functional activity? o = the amount needed to digest bacteriophage lambda DNA completely in one hour How to join pieces of DNA together • Many RE cut palindrome DNA sites (with two identical interfaces) o 5’ TATA 3’ – palindrome ▪ 3’ ATAT 5’ – palindrome • A palindromic sequence is a sequence made up of nucleic acids within double helix of DNA and/or RNA that is the same when read from 5' to 3' on one strand and
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