chapter 10

Question 1

recombinant DNA technology (RDT)

Answer 1

methods for obtaining, amplifying, and manipulating DNA fragments

Question 2

how are DNA molecules cut

Answer 2

by restriction enzymes at the cut site to generate sticky or blunt ends that are later used for DNA insertion into the plasmid

Question 3

restriction enzymes (REs)

Answer 3

cut DNA strands by breaking covalent (phosphodiester) bonds of backbone

Question 4

recognition sites

Answer 4

specific DNA sequences that REs bind to (usually downstream to promoter region)

Question 5

cut sites

Answer 5

where REs cut DNA

Question 6

sticky (overhang) ends

Answer 6

single strand with complementary base pairs, 5’ overhand

Question 7

blunt ends

Answer 7

have no overhand

Question 8

EcoR1

Answer 8

restriction enzyme in e.coli that generates sticky ends

Question 9

palindromic sequence

Answer 9

cut sites are usually palindromic, most recognized by RE

Question 10

why did REs evolve (3 reasons)

Answer 10

1. occur naturally in many prokaryotes
2. defense against phages
3. modify their own DNA to prevent self digestion

Question 11

joining the DNA molecules

Answer 11

DNA ligase joins the inserted DNA into the vector/plasmid

Question 12

DNA ligase

Answer 12

joins two DNA molecules by covalent linkage of deoxyribose backbone (can join blunt with sticky ends if there’s a 5’ phosphate and 3’ OH in the gap)

Question 13

insertion of donor DNA into the plasmid (3)

Answer 13

1) Donor and plasmid DNA are cut with compatible REs (i.e their ends are compatible)
2) sticky ends hybridize because of base pairing but there’s a gap in backbone (no phosphodiester bond)
3) ligase seals gap in backbone

Question 14

replication and amplification of DNA

Answer 14

recombinant plasmid DNA is introduced into host cells through transformation where it’s replicated

Question 15

amplification’s 2 mechanisms

Answer 15

1) all cells in culture have 1 copy of plasmid
2) many plasmids maintained in multiple copies per cell

Question 16

only circular DNA molecules replicated ____, linear DNA must ____

Answer 16

1) autonomously
2) integrate into host chromosome

Question 17

isolating specific sequence approaches (2)

Answer 17

library method and PCR amplification of specific sequence

Question 18

library method

Answer 18

DNA fragments carried on plasmids or in phages are amplified by e.coli and screened for desired sequence

Question 19

PCR amp of specific sequence

Answer 19

DNA polymerase used with specific primers to synthesize specific fragments

Question 20

cloning

Answer 20

isolating and amplifying particular sequence (finding sequences corresponding to specific gene and amplify by plasmid insertion)

Question 21

cloning with library method

Answer 21

break genome into small gene sized pieces (creating LIBRARY in cloning vector) and identify piece of interest with detection method

Question 22

cloning vector

Answer 22

carries foriegn pieces of DNA to be replicated and amplified in cell systems

Question 23

4 basic cloning vectors

Answer 23

plasmids, lambda phage, fosmids, and artificial chromosomes

Question 24

plasmid vectors

Answer 24

most convenient, small, easy manipulation

Question 25

bacteriophage vectors

Answer 25

allows larger inserts, easy to infect into bacteria and high level protein production

Question 26

fosmid and artificial chromosomes (YACs and BACs)

Answer 26

can carry largest inserts

Question 27

all plasmids have (4)

Answer 27

- origin of replication (determines copy number) - polylinker/MCS (multiple cloning vectors with unique cut sites) - selectable marker (in case transformation is insufficient) - means to detect inserts (in case ligase insufficient)

Question 28

types of libraries (2)

Answer 28

genomic fragments and cDNA

Question 29

genomic fragments

Answer 29

made by randomly breaking genome, represents entire genome

Question 30

cDNA

Answer 30

each vector has single gene or gene frag, ONLY protein coding sequences (unequal rep)

Question 31

making genomic library (3)

Answer 31

1. digest genomic DNA and plasmid vector with same RE 2. mix digested plasmid and genomic DNA (joined with ligase) 3. transform into e.coli

Question 32

making cDNA (5)

Answer 32

1) lyse cells and extract total RNA 2) putify mRNA using olig(df) column 3) use reverse transcriptase + olig(df) primer to make SS cDNA 4) use oligonucleotide primer and DNA polymerase II to synthesize complementary strand 5) ligate resulting duplex cDNAs into cloning vector

Question 33

cloning methods

Answer 33

probes, complementation

Question 34

probes

Answer 34

molecules designed to recognize library vectors with specific sequence on basis of structure