Lectures 1&2: DNA Technology I&II

Question 1

What type of sequences do restriction enzymes recognize?

Answer 1

Palindromic sequences

Question 2

What are palindromic sequences?

Answer 2

when the bases are read 5’ –> 3’, the sequence is the same on both strands of DNA
ex. 5’-GGATCC-3’

Question 3

What is a Restriction Map used for?

Answer 3

identify Restriction Fragment Length Polymorphisms

Question 4

What is a Restriction Fragment Length Polymorphism?

Answer 4

sequence differences between human individuals

Question 5

What are 5’ or 3’ overhangs used for, and how are they created?

Answer 5

Used for DNA cloning

Created by restriction enzymes

Question 6

Besides transferring the DNA to nitrocellulose or nylon fiber, what happens during the transfer step of southern blotting? Why is this important?

Answer 6

strong base (high pH) denatures DNA –> can be hybridized

Question 7

What label is usually used in a DNA probe?

Answer 7

32-P

Question 8

what is the most effective end to leave by a restriction enzyme?

Answer 8

sticky end with complementary sequence to ligating sequence

Question 9

What enzymes are used to cut DNA for the purposes of cloning?

Answer 9

Restriction endonucleases

Question 10

What enzyme connects sticky or blunt ends to other DNA molecules? Where is it from?

Answer 10

T4 DNA lygase

Bacteriophage

Question 11

What does T4 DNA ligase use for energy?

Answer 11

ATP

Question 12

What type of vector can carry the largest amount of exogenous DNA? Where are they found? What type of cloning do they do?

Answer 12

Yeast artificial plasmid (100-2000kb)

yeast, genomic

Question 13

What type of vector can carry the smallest amount of exogenous DNA? Where are they found? What type of cloning do they do?

Answer 13

Plasmid (15kb)
Bacteria, yeast
genomic, cDNA

Question 14

what is cDNA? how is it produced?

Answer 14

dsDNA copies of mRNA

reverse transcriptase

Question 15

What is the downfall to genomic libraries?

Answer 15

Most material is non-coding (~1% becomes final mRNA)

Question 16

What is the potential downfall to cDNA libraries?

Answer 16

selected tissue might not express gene of interest to a large degree

Question 17

For what does nitrocellulose paper have high affinity?

Answer 17

DNA

Question 18

What is the formula for the amplification of PCR?

Answer 18

starting templates x 2^# cycles

Question 19

What does Northern blotting isolate?

Answer 19

RNA

Question 20

What does Southern blotting isolate?

Answer 20

DNA

Question 21

What 3 things does Northern blotting tell you

Answer 21

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

Question 22

In what DNA would Northern blotting reveal that mRNA for a given gene is not the correct size?

Answer 22

Muscular dystrophy

dystrophin gene is smaller than normal due to exon deletion

Question 23

What is a protein’s isoelectric point?

Answer 23

pH at which it has no net charge

Question 24

what two elements are present in 2-D gel electrophoresis?

Answer 24

Isoelectric focusing

denaturing

Question 25

What does Western blotting look at?

Answer 25

Proteins

Question 26

What enzyme is commonly coupled to secondary antibodies in Western blotting?

Answer 26

Horseradish Peroxidase (HRP)

Question 27

What two things can Western blotting tell you about a protein?

Answer 27

if it is expressed

if it is the proper size

Question 28

What is immuno-fluorescence microscopy of proteins used for?

Answer 28

cellular expression

cellular localization

Question 29

What two functions does Dicer have in RNAi?

Answer 29

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

Question 30

What type of enzyme is Dicer?

Answer 30

RNA endonuclease

Question 31

What enzyme works with Dicer?

Answer 31

RNA helicase