chapter twenty part one

Question 1

restriction endonucleases

Answer 1

discovered late 1960s, isolated from bacteria
- cut DNA molecules at limited # of specific locations

Question 2

function of restriction enzymes in bacteria

Answer 2

protect bacterial cell by cutting up foreign DNA from other organisms/phages

Question 3

how do restriction enzymes know not to cut up the bacteria’s own DNA?

Answer 3

bacteria sues modifying enzymes to label own DNA, like methyl groups

Question 4

where do restriction enzymes cut

Answer 4

restriction site - particular short DNA sequence, typically symmetrical

Question 5

what do restriction enzymes leave?

Answer 5

sticky ends
- can produce various combinations
- available for H-bonding w/ complementary bases of other sticky end

Question 6

what seals the sticky ends together?

Answer 6

DNA ligase

Question 7

how many restriction enzymes are there

Answer 7

hundreds

Question 8

name of restriction enzymes

Answer 8

EcoR1
- E - genus
- co - species
- strain
- 1 - sequence #

Question 9

recombinant DNA

Answer 9

contains DNA from 2 dif sources (plasmid and donor)

Question 10

plasmid

Answer 10

small, circular DNA molecules that act as cloning vector

Question 11

procedure of DNA cloning with restriction enzymes

Answer 11

1. human DNA (donor) and plasmid (bacteria) extracted
2. both cut w/ same restriction endonuclease
3. sticky ends joined by chance - recombinant DNA, seals with. ligase
4. bacteria take up recombinant DNA by transformation
5. population (colony) of bacteria will grow and be clones of each other
6. sift through clones (library) to find clone of interest, proteins produced harvested

Question 12

DNA libraries

Answer 12

collections of recombinant DNA molecules
- typically in host cell

Question 13

genomic library

Answer 13

has copies of all genes from a genome

Question 14

cDNA library

Answer 14

• from particular tissue - smaller library

Question 15

cDNA

Answer 15

complementary DNA
- made from reverse transcribing mRNA

Question 16

gel electrophoresis

Answer 16

uses gel made of polymer that has microscopic holes of dif sizes through which shorter fragments can travel faster

Question 17

what does gel electrophoresis separate DNA molecules by?

Answer 17

charge and size

Question 18

if molecules have an equal charge/mass ratio (like DNA), then molecules can be separated by

Answer 18

length (size) alone

Question 19

amplification of DNA or RNA without cloning

Answer 19

only work with small region of interest, fast

Question 20

polymerase chain reaction discovery

Answer 20

1983, Kary Mullis
- Nobel prize 1993

Question 21

PCR steps

Answer 21

1. mixture heated to denature/separate strands of DNA
2. called to allow H-bonding of short complementary ssDNA primers
3. dNA poly extends primers 5’ to 3’
   process repeated

Question 22

PCR overview

Answer 22

• produces lots of DNA from a small segment of DNA quickly
• billions of copies in a few hours

Question 23

RT-PCR

Answer 23

reverse transcriptase-polymerase chain reaction
- uses rNA as template to make dsDNA (cDNA), then PCR occurs

Question 24

DNA sequencing

Answer 24

using complementary base pairing to determine complete nucleotide sequence of DNA molecule

Question 25

manual DNA sequencing

Answer 25

about 1000 nucleotide pairs/day since about 1980

Question 26

current automated DNA sequencing

Answer 26

about 900 million nucleotides in 10 hours
- 1000 npt per second

Question 27

time/cost to sequence your genome

Answer 27

6 hours, $1,000

Question 28

what do we now have genomes sequenced of?

Answer 28

• living and extinct organisms
• over 4,000 bacterial species
• almost 200 eukaryotic species