lecture 17

Question 1

recombinant DNA technology

Answer 1

ability to manipulate DNA

Question 2

REs (restriction endonucleases)

Answer 2

“DNA scissors” that cleave DNA at specific sequences

most recognition sequences are 4-6 bp long and palindromic

cuts can be blunt or leave a sticky overhang

Question 3

gel electrophoresis is used to

Answer 3

separate DNA of different sizes

Question 4

hybridization is the process of

Answer 4

DNA renaturation

Question 5

how does hybridization work?

Answer 5

increasing temp can denature the DNA to release single strands

lowering temp can cause strands to renature

Question 6

southern blotting

Answer 6

the transfer of DNA to a membrane and hybridization w a labeled probe

Question 7

northern blotting

Answer 7

same process as southern blotting but w RNA

Question 8

DNA cloning

Answer 8

producing many identical copies of a DNA sequence

Question 9

recombinant DNA

Answer 9

DNA molecule w DNA from many sources

Question 10

plasmid

Answer 10

small circular DNA molecule used in bacteria

Question 11

plasmids can be introduced into bacteria through

Answer 11

transformation

Question 12

when the bacteria has taken up the plasmid, when they replicate..

Answer 12

the plasmid is also replicated so you end up w a lot of copies

Question 13

genomic library

Answer 13

representative of all the genomic sequence of an organism

-includes coding and noncoding DNA
-library will be the same regardless of cell type

Question 14

cDNA library only contains

Answer 14

genes transcribed into mRNA

Question 15

4 steps to make recombinant DNA

Answer 15

1. cut the DNA of interest
2. cut the plasmid vector
3. insert DNA into the plasmid
4. reseal nicks w DNA ligase

Question 16

3 steps to create a genomic library

Answer 16

1. the genome is digested using REs or DNA shearing
2. the DNA fragments are cloned into plasmids
3. plasmids are introduced into E coli as hosts

Question 17

5 steps to create a cDNA library

Answer 17

1. total RNA is extracted from organism, isolate mRNA
2. cDNA (complementary) is prepped using reverse transcriptase using a polyT primer complementary to the polyA tail of mRNA
3. the cDNA is inserted into a vector (plasmid) and cloned to produce a cDNA library
4. cDNA can be inserted into a vector
5. introduce vector to bacteria

Question 18

PCR

Answer 18

polymerase chain reactor

= a very powerful technique, allows you to make billions of copies of a nucleic acid

v sensitive, fast and easy

Question 19

3 steps for PCR

Answer 19

1. heat to separate strands
2. cool and anneal primers that flank the sequence of interest
3. allow DNA polymerase to extend from the primer

***this is repeated 30-35 times!

Question 20

why do we need a thermostable DNA polymerase for PCR?

Answer 20

if it wasn’t, the DNA polymerase would denature when heated!
we would need to add a fresh one after each cycle!

Question 20

PCR uses

Answer 20

• used while generating DNA libraries to amplify segments being cloned
• used to detect small amounts of DNA/RNA from a pathogen
• can be used to amplify STRs to be used in DNA fingerprinting

Question 21

sanger sequencing makes use of

Answer 21

ddNTPs (dideoxyribonucleoside triphosphates)

Question 22

why is DNA polymerization impossible is a ddNTP is incorporated?

Answer 22

ddNTPs have a 3’ H instead of a 3’ OH which makes DNA polymerization impossible

Question 23

DNA microarray

Answer 23

a slide has hundreds of DNA fragments in diff spots that are complementary to mRNA for diff genes

Question 24

RNA seq

Answer 24

RNA from a cell is isolated and then sequenced

Question 25

what is the advantage of RNA seq over microarray?

Answer 25

don’t need to know sequence of mRNA ahead of time for RNA seq

Question 26

in situ hybridization uses a

Answer 26

labeled single strand DNA or RNA probe that targets a specific sequence in a cell

Question 27

a gene encoding a protein of interest is fused to a

Answer 27

reporter gene

Question 28

reporter gene=

Answer 28

a gene that encodes a protein product that can be easily modified

Question 29

gene knockout

Answer 29

completely eliminating a gene leads to a gene knockout which can be used to simulate disease conditions