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Restriction Enzyme Analysis
Based on slides presented by Dr. DeJoung at the University of Texas at Dallas
18
Biochemistry
Undergraduate 3
10/12/2011

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Term
The real purpose of restriction enzymes is _____________________
Definition
to protect bacteria from invading DNA
Term
Restriction enzymes have been very important to _________
Definition
manipulate, move, and propagate pieces of DNA in E.coli using plasmid cloning vectors.
Term
Why don't bacteria digest their own DNA with restriction enzymes?
Definition
Restriction enzymes typically come from two genes or
activities, a restriction activity and a methylase activity.
Methylation of DNA bases at the recognition sequence
prevent the RE from cutting. Since entering
bacteriophage DNA is not methylated, it is cut.
Term
Type II and Type II enzymes cleave DNA chains at ________.
Definition
specific recognition sequences
Term
How many bases do enzymes usually recognize?
Definition
4,6, or more
Term
How much ATP is required for digestion?
Definition
no ATP is needed
Term
EcoRI names come from
Definition

E=E.

co = coli

RI = 1st restriction enzyme found in R strain of E. coli

Term
EcoRI sequence recognized
Definition
G|AATT C
C TTAA|G
Term
If you start with a complex DNA, like human genomic DNA isolated from cells, the digestion products will _____ the gel.
Definition
appear as a smear from top to bottom of
Term
Isoschizomers
Definition
pairs of restriction enzymes specific to the same recognition sequence
Term
Sanger
Definition
developed the dideoxy method and found the aa sequence for insulin (2 nobel prizes)
Term
dideoxynucleotide analogs
Definition
nucleotide analogs used as chain terminators to determine sequence of DNA
They possess no 3'-OH. Instead they have a 3'-H that cannot be used for elongation.
Term
dideoxynucleotide electrophoresis sequence is read ____________________
Definition
from the bottom of the gel to the top -> this is the complement of the template.
Term
454 Sequencing
Definition
Template DNA molecules are immobilized on microscopic beads. Hundreds of thousands to millions of beads are analyzed at once so that sequencing of many different DNAs are performed and
read at the same time.
Term
pyrophosphate
Definition
This is released whena nucleotide is correctly added to the end of one of
the DNA templates. This is used in 454 Sequencing.
Term
What enzymes work with pyrophosphate to produce light?
Definition
ATP sulfurylase and luciferase
Term
Reaction catalyzed by ATP sulfurylase
Definition
PPi + APS → ATP + SO42- (used in conversion of PPi to light)
Term
Reaction catalyzed luciferase
Definition
ATP + luciferin + O2 → AMP + CO2 + oxyluciferin + light
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