Pharmocology Test 2

Front 1

What are the four essential components of DNA cloning?

Back 1

1 isolating and joining segmnts of DNA

2 insertion of the recombinant DNA into a self-replicating plasmid vector

3 introduction of the vector containing the recombinant DNA into a recipient cell

4 selecting the cells which have acquired the recombinant DNA

Front 2

What type of vectors are used for creating recombinant dna?

Back 2

Plasmids - double stranded circular molecules of DNA that exist independent of the bacterial chromosome

Front 3

what are plasmids?

Back 3

double stranded circular molecules of DNA that exist independent of bacterial chromosome

common and widespread in prokaryotes.

Front 4

what is the only eukaryote that harbors plasmids

Back 4

yeast

Front 5

what is a copy number?

Back 5

number of plasmids found per cell in a normal bacteria.

note: higher the copy number the better the recovery of recombinant DNA.

Front 6

Why use antibiotic resistant genes?

Back 6

enables growth in presence of toxic concentration of certain antibiotics.

useful for selection of cells which harbor the plasmids since they will be resistant to antibiotic present in the medium.

most common forms of resistance: ampicillin, hygromycin B, tetracycline, and kanamycin

Front 7

Plasmid Components: What is the origin?

Back 7

DNA sequence that initiates replication

enables plasmid to produce multiple copies of itself and any recombinant DNA inserted into the plasmid

Front 8

Plasmid Components: What are MCS's (multipule cloning sites)?

Back 8

engineered segment of DNA which contains recognition sequences for multiple restrictino endonucleases over a short stretch of nucleotide bases.

MCS makes the insertion of a foreign DNA sequence into a specific region of the vector much more convenient.

Front 9

What do nucleases do?

Back 9

cut, sorten and degrade DNA and RNA

Front 10

What do Ligases do?

Back 10

join nucleotides together by creating phosphodiester bonds

Front 11

What do polymerases do?

Back 11

create complementary nucleotide chain to an existing DNA or RNA sequence

Front 12

What do exonucleases do?

Back 12

1) cleave nucleotides one at a time from the end of DNA molecules

2) may be specific for starting at either the 3' end or the 5' end

Front 13

What do Endonucleases do?

Back 13

cleave DNA at internal phosphodiester bonds

Front 14

What happens when a nuclease cleaves a phosphodiester bond?

Back 14

the nuclease cleaves the phosphodiester bond of adjoining nucleotides. this cleavage disrupts hydrogen bonding between base pairs of dsDNA

Front 15

what do restriction endonucleases do? give specific example...

Back 15

recongnize specific palindromic sequences within DNA and will cut the DNA into various size fragments

AA/TT
TT/AA

Front 16

how do restriction endonucleases determine size and number?

Back 16

dependent solely upon where and how frequent the recognition sequences are given.

1 recognition site = 2 fragments

4 recognition sites = 5 fragments

circular dna - 1 rec site - 1 fragment

Front 17

what are the two ends restriction endonucleases can produce when cutting dna?

Back 17

blunt and sticky ends

Front 18

DNA ligases do what?>

Back 18

repair breaks in phosphodiester bonds

work on sticky or blunt ends

Front 19

what does DNA Polymerase holoenzyme do?

Back 19

contains 5' exonuclease activity which degrades the DNA while polymerase activity of the enzye replaces the degraded base with a new base

SEE SLIDE p 9

Front 20

What does a Klenow Fragment of DNA polymerase do?

Back 20

lacks 5' exonuclease activity and therefore is more oftern used to fill in gaps in dsDNA

SEE SLIDE p 9

Front 21

What does a reverse transcriptse do?

Back 21

uses RNA to produce a DNA compliment (cDNA) and then cell procedes through transcription/translation

Front 22

in Electrophoresis what does agarose gel do?

Back 22

serves as sieve through which DNA fragments migrate in response to application of an electric current

DNA has overal negative charge so the fragments migrage from - pole to + pole

Front 23

what is ethidium bromide?

Back 23

non specific dye

intercalates between DNA bases and fluoresces upon exposure to ultra-violet light

will detect all DNA in gel regardless of sequence

Front 24

hybridization uses what kind of probes?

Back 24

oligonucleotide probes

Front 25

what are steps of hybridization of DNA?

Back 25

transfer or blot the DNA present in agarose gel onto a special filter paper called nitrocellulose.

interaction of the nucleotides wiht the membrane is then stabilized by crosslinking the sugar phosphat backbone of th DNA to the membrane with heat or UV light

the nitrocelulose membrane is then incubated with a radioactive DNA probe that is complementary to our gene of interest to allow anealing to the complementary sequence present in DNA bound to membreane.

the radioactive probe is removed and unbound

detect position of dna on the membrane by exposing the blot to film, AUTORADIOGRAPHY

Front 26

southern blots are used for?

Back 26

detecting specific nucleic sequences for DNA:DNA hybridization

Front 27

northern blots are used for?

Back 27

RNA:DNA or RNA:RNA hybridization

Front 28

When designing probes what are three ways or knowlege of structure to help design?

Back 28

1) having a partial sequence of the gene

2) having a full or partial sequence of a closely or even distantly related gene

3) knowing the protein sequence of the gene product

Front 29

When using an oligonucleotide probe based on protein sequence what information do you need?

Back 29

1) partial sequence of the protein which we want to clone

Front 30

what is the main difficulty using an oligonucleotide probe?

Back 30

the degeneracy of some probes. many protiens can be coded by numerous codons

it is best to use streches of amino acids in the proteins that are only coded by one codon.