PREPARATION OF DNA AND RNA + THE ENZYMES

Question 1

what are types of material that we extract from cells

Answer 1

genomic DNA or ADN and plasmid DNA

Question 2

genomic DNA is… plasmid DNA is…

Answer 2

-associated with proteins
-nude

Question 3

what do you we use to extract DNA for the organic method ? (chemicals)

Answer 3

phenol and chloroform

Question 4

we use phenol for… and chloroform for…

Answer 4

-dissolve proteins
-dissolve lipids

Question 5

and H2O for…

Answer 5

dissolve nucleic acids, salts, sugar

Question 6

what are the steps of organic method of extraction ?

Answer 6

-Homogenization in denaturing conditions
-separation by centrifugation
-RNA/DNA treatment
-collection

Question 7

on what do we use the organic method ?

Answer 7

cells or organs

Question 8

we us… to extract DNA during the separation

Answer 8

RNAase

Question 9

we can we use instead of phenol

Answer 9

trizol

Question 10

steps for the silica method

Answer 10

-homogenization
-DNA binding
-Elution of DNA

Question 11

in the silica homogenization we add… (4) to lyse de cells

Answer 11

-detergent
-proteinase K
-DNAase or RNAase

Question 12

after the silica homogenization we… to obtain pure nucleic acids (DNA binding)

Answer 12

wash with water other molecules

Question 13

Silica, DNA and RNA are… charged so there’s a need to equilibrate charges with an…. to make them interact

Answer 13

• negatively charged
• increase of ion power

Question 14

the… column is… so the DNA will… and go through…

Answer 14

• cationic
• • charged
• be attracted to it
  -silica

Question 15

steps for the preparation of plasmid DNA

Answer 15

-alkaline lysis
-homogenization
-isolation of the plasmid

Question 16

alkaline lysis : NaOH to … SDS to… and RNAase to..

Answer 16

-break H bonds
-solubilize H bonds
-solubilize RNA

Question 17

what do we use in homegenization

Answer 17

-K+ acetate to renature only the plasmid
-phenol cholorform centrifugation

Question 18

who do we isolate the plasmid ?

Answer 18

ethanol

Question 19

what are the two types of restrictions enzyme ?

Answer 19

restriction endonulease and restriction methylases

Question 20

what are the 2 types of nucleases

Answer 20

-exonucleases = break the first or last bound
-endonucleases = break within the DNA

Question 21

endonuclease type 1 = … at 1kb (…)

Answer 21

• cleavage
• 1000 base paris

Question 22

endonuclease type 2 : cleavage within…

Answer 22

restriction site

Question 23

endonuclease type 3

Answer 23

cleavage at 10 pb

Question 24

the restriction is …, … and is recognized with … nucleotides

Answer 24

-palindromic
-specific to one enzyme/bacteria
-4-6

Question 25

types of cleavage

Answer 25

blunt or sticky ends

Question 26

blunt ends = …. and are …, …

Answer 26

• Pvu II
• compatible
• can be reassociated with

Question 27

sticky ends are … they can… so they are…

Answer 27

-complementary
-hybridize
-cohersive

Question 28

types of sticky ends ?

Answer 28

• EcoR1
• Pst1

Question 29

5’ overhangs

Answer 29

EcoR1

Question 30

3’ overhangs

Answer 30

Pst1

Question 31

in which case the intergration is oriented

Answer 31

• 1 sticky end + 1 blunt end with a different enzyme
• 2 sticky ends with different enzyme

Question 32

2 blunts ends with same enzyme

Answer 32

compatible, non complementary, non oriented

Question 33

2 blunts ends with different enzyme

Answer 33

• compatible
• non complementary
• non oriented

Question 34

2 sticky ends with same enzyme =

Answer 34

• compatible
• complementary
• non oriented

Question 35

2 sticky ends with different enzyme

Answer 35

• non compatible
• non complementary
• oriented

Question 36

1 blunt + 1 sticky with different enzymes

Answer 36

• non compatible
• non complementary
• oriented

Question 37

pancreatic DNAase I is a… and makes

Answer 37

• Other DNA endonuclease
• non specific cleavage

Question 38

nuclease S1 (action + cible)

Answer 38

-digest sticky ends to produce blunts ends
-single strand DNA or RNA

Question 39

RNAse H (action + cible)

Answer 39

• digest DNA heteroduplex
• the DNA strand

Question 40

what do we need to synthesize DNA (4)

Answer 40

• DNA polymerase
• dNTP
• template
• primer (DNA or RNA with free 3’OH end)

Question 41

what enzymes work in the 5’-3’ direction ?

Answer 41

polymerase and bacterial exonuclease

Question 42

exonuclease

Answer 42

proofreading

Question 43

the polymerase we use in vitro is … we use in vitro to ensure the removing of the primer thanks to …. the fragment we obtain is called …

Answer 43

• polymerase I modified
• a toxin named subtilsin
• Klenow DNA pol

Question 44

thermo resistant DNA pol (2)

Answer 44

• Taq DNA pol
  -Pfu DAN pol

Question 45

what do we need to synthesize RNA (4)

Answer 45

• RNA pol
• NTP
• template
• Promotor (DNA)

Question 46

RNA pol for bacteria

Answer 46

RNA pol I (5 sub)

Question 47

RNA pol for eukaryotes (3)

Answer 47

RNA pol I, II, III (12 sub at least)

Question 48

others activity of the RNA pol (4)

Answer 48

• phosphatase
• polynucleotide-kinase
• ligase
• topoisomerase

Question 49

phosphatase

Answer 49

cleave the phosphate from 5’ nucleotide

Question 50

polynucleotide-kinase

Answer 50

incorporate a a phosphate

Question 51

ligase

Answer 51

create phosphor-ester bonds to recover DNA

Question 52

topoisomerase

Answer 52

cleave and changes to form