Exam Two: Ch 13 Learning Objectives

Question 1

What is the role of restriction enzymes?

Answer 1

cleave DNA at specific sequence recognition sites

Question 2

T/F: restriction enzymes only cleave at non-modified DNA

Answer 2

true

Question 3

Where do Type II restriction enzymes cleave compared to Types I and III

Answer 3

II: cleave within 4-8 base sequence recognized by corresponding modification methylase
I and III: other sites

Question 4

Type II restriction enzymes can also cleave ___ DNA sequences

Answer 4

palindromic

Question 5

Where does TaqI cleave?

Answer 5

T(cleave)CGA

Question 6

What is the recognition sequence for BamHI?

Answer 6

5’G(cleave)GATC

Question 7

What is the recognition sequence for EcoRI?

Answer 7

5’G(cleave)AATTC

Question 8

What is the recognition site for PstI?

Answer 8

5’CTGCA(cleave)G

Question 9

What is the recognition site for PvuII?

Answer 9

5’CAG(cleave)CTG

Question 10

What is the difference between sticky and blunt ends?

Answer 10

sticky ends cleave asymmetrically
blunt ends cleave symmetrically

Question 11

What is the role of DNA ligase?

Answer 11

enzymes that can from a phosphodiester bond at a single-strand break in DNA

Question 12

What are the 5 steps to creating Recombinant DNA?

Answer 12

Cut source DNA at boundaries of genes
Select vector (carrier DNa)
Insert gene into vector
Insert recombinant vector into host cell
Host produces multiple copies of recomb DNA

Question 13

What is one advantage to Plasmid DNA?

Answer 13

replicates easily

Question 14

How many base pairs does pBR322 have?

Answer 14

4361

Question 15

What are the 5 restriction sites found at pBR322?

Answer 15

PtsI (ampicillian resistant) EcoRI, BamHI + SaII (= Tetracycline resistant), PvuII and origin of replication

Question 16

pBR322 is commonly used as a ?

Answer 16

vector

Question 17

How does lacZ work?

Answer 17

encodes beta-galactosidases, which cleaves a colorless compound into a blue product

Question 18

Where is lacZ found?

Answer 18

pUC18

Question 19

What are the three steps to PCR?

Answer 19

denaturing, annealing, and extension

Question 20

Describe the denaturing step of PCR

Answer 20

heating strands at 94C; separates strands

Question 21

Describe the annealing step of PCR

Answer 21

add forward and reverse primers at 45-65C

Question 22

Describe the extension step of PCR also known as Sanger method

Answer 22

primer elongation by thermophillic DNA polymerase at 74C in the presence of dATP, dGTP, dCTP, and dTTP

Question 23

Roughly explain the Sanger method

Answer 23

incoming nucleotides added as deoxy at 3’
catalyzed by DNA polymerase
5’ primer to 3’ template strand

Question 24

Chain termination method is also known as

Answer 24

Sanger method

Question 25

What is the function of the Sanger method?

Answer 25

method of determining the nucleotide sequence of DNA