Lectures 1&2: DNA Technology I&II Flashcards
What type of sequences do restriction enzymes recognize?
Palindromic sequences
What are palindromic sequences?
when the bases are read 5’ –> 3’, the sequence is the same on both strands of DNA
ex. 5’-GGATCC-3’
What is a Restriction Map used for?
identify Restriction Fragment Length Polymorphisms
What is a Restriction Fragment Length Polymorphism?
sequence differences between human individuals
What are 5’ or 3’ overhangs used for, and how are they created?
Used for DNA cloning
Created by restriction enzymes
Besides transferring the DNA to nitrocellulose or nylon fiber, what happens during the transfer step of southern blotting? Why is this important?
strong base (high pH) denatures DNA –> can be hybridized
What label is usually used in a DNA probe?
32-P
what is the most effective end to leave by a restriction enzyme?
sticky end with complementary sequence to ligating sequence
What enzymes are used to cut DNA for the purposes of cloning?
Restriction endonucleases
What enzyme connects sticky or blunt ends to other DNA molecules? Where is it from?
T4 DNA lygase
Bacteriophage
What does T4 DNA ligase use for energy?
ATP
What type of vector can carry the largest amount of exogenous DNA? Where are they found? What type of cloning do they do?
Yeast artificial plasmid (100-2000kb)
yeast, genomic
What type of vector can carry the smallest amount of exogenous DNA? Where are they found? What type of cloning do they do?
Plasmid (15kb)
Bacteria, yeast
genomic, cDNA
what is cDNA? how is it produced?
dsDNA copies of mRNA
reverse transcriptase
What is the downfall to genomic libraries?
Most material is non-coding (~1% becomes final mRNA)
What is the potential downfall to cDNA libraries?
selected tissue might not express gene of interest to a large degree
For what does nitrocellulose paper have high affinity?
DNA
What is the formula for the amplification of PCR?
starting templates x 2^# cycles
What does Northern blotting isolate?
RNA
What does Southern blotting isolate?
DNA
What 3 things does Northern blotting tell you
whether mRNA for a gene is present in collected cells/tissue/patient
expression level of mRNA for that gene
whether mRNA for that gene is the correct size
In what DNA would Northern blotting reveal that mRNA for a given gene is not the correct size?
Muscular dystrophy
dystrophin gene is smaller than normal due to exon deletion
What is a protein’s isoelectric point?
pH at which it has no net charge
what two elements are present in 2-D gel electrophoresis?
Isoelectric focusing
denaturing
What does Western blotting look at?
Proteins
What enzyme is commonly coupled to secondary antibodies in Western blotting?
Horseradish Peroxidase (HRP)
What two things can Western blotting tell you about a protein?
if it is expressed
if it is the proper size
What is immuno-fluorescence microscopy of proteins used for?
cellular expression
cellular localization
What two functions does Dicer have in RNAi?
Cleaves dsRNA introduced to cell into active ~23kb pieces
Once these pieces bind to complementary sequences, Dicer cuts these sequences, which are then further cut by exonucleases preventing them from being translated
What type of enzyme is Dicer?
RNA endonuclease
What enzyme works with Dicer?
RNA helicase
