Lecture #1 - Basic Techniques

Question 1

Genetic engineering

Answer 1

Ability to manipulate DNA with precision

Question 2

Recombinant DNA technology

Answer 2

Combining two different DNA

Question 3

Key Techniques of recombinant DNA technology

Answer 3

1. Cutting of specific sites of DNA via restriction nucleases
2. DNA ligation
3. DNA cloning
4. Nucleic acid hybridization
5. Rapid sequencing of DNA nucleotides
6. DNA microarrays

Question 4

DNA ligation

Answer 4

Joining DNA molecules via DNA ligase

Question 5

DNA ligase catalyzes formation of _______.

Answer 5

phosphodiester bonds

Question 6

DNA cloning

Answer 6

Many copies via cloning vectors or PCR

Question 7

Nucleic acid hybridization

Answer 7

Uses complementary base pairing to find specific DNA or RNA sequences

Very accurate

Question 8

Rapid sequencing of DNA nucleotides

Answer 8

Makes it possible to identify genes and deduce amino acid sequence of the protein they encode

Question 9

DNA microarrays

Answer 9

monitor mRNA levels of every single gene in the cell

Question 10

Restriction nucleases

Answer 10

enzymes purified from bacteria that degrade viral DNA
Cut DNA at specific sites 4-8 nucleotides long
Palindromic cutting

Question 11

Staggered cuts

Answer 11

Restriction nucleases leave overhangs or “sticky ends” during DNA restriction
Easy to hybridize

Question 12

Gel electrophoresis (GE)

Answer 12

Separates DNA molecules of different sizes

Determines length and purity of DNA molecules

Question 13

Nucleotides have a _______ charge, which makes GE easy since they move naturally to the _______ end of the chamber.

Answer 13

negative; positive

Question 14

DNA bands are made visible by staining/labeling DNA with _______.

Answer 14

ethidium bromide (fluorescent dye)

Question 15

Less agarose means _______ pores.

Answer 15

larger

Question 16

Bigger DNAs move _______ on GE due to pores.

Answer 16

slowly and not as far

Question 17

What technique would you use to separate very small molecules?

Answer 17

PAGE polyacrylamide gel electrophoresis

Question 18

Pulsed-field GE

Answer 18

Separates extremely long DNA molecules
They change direction in the field
Larger molecules take longer to change directions

Question 19

Purified DNA molecules can be labeled with _______ or _______. An example is _______.

Answer 19

chemical markers; radioisotopes; 32P

Question 20

What are the two ways to label DNA? Which gives a stronger signal and why?

Answer 20

1. DNA polymerase copies DNA in presence of a nt labeled with 32P; stronger as it labels every nt
2. Bacteriophage enzyme polynucleotide kinase labels the 5’ end of each chain with 32P; weaker as it only labels the 5’ ends

Question 21

DNA denaturation

Answer 21

High temp. (100C) separates base pairs into two strands

Question 22

DNA renaturation

Answer 22

Lower temp. (65C) reforms double helix via rehybridization

Question 23

DNA probe

Answer 23

ss DNA used to detect complementary sequences
Carry radioactive or chemical markers
Gives an exact match at stringent conditions (42C)
Can be used to find similar, not exact genes at non stringent conditions (35C)

Question 24

Southern blot

Answer 24

Detection of DNA

Question 25

Northern blot

Answer 25

Detection of RNA

Question 26

Western blot

Answer 26

Detection of protein