Chp 2,2: ADN recombinant et ADN vectoriel

Question 1

2 types de vecteurs les plus fréquemment utilisés

Answer 1

• bactériophages (virus bactérien à ADN circulaire, not all of them are db)
• plasmides (ADNdb circulaire)

Question 2

how do ADN ligase ligate dna fragments

Answer 2

-during dna replication, the ligase catalyse the reunion of ends of short fragments of Okazaki

• ADN vectoriel and the fragments de restrictions sont liées de façon covalence par des liaisons phosphodiester 3’->5’
• on top of ligating the sticky ends complémentaire, the ADN ligase of a bactérophage T4 can ligate whatever pairs at the blunt ends of DNA.
• the 3’-hydroxyle group and 5’-phosphate group ligate by ADN ligase of T4

-ADN ligase NEEDS ATP for the ligation

Question 3

clonage directionnel?

Answer 3

• directional cloning is when the DNA fragment is inserted into the vector in a specific orientation (rather than the fragment being inserted is flipped)

Question 4

do not get carte génétique mixed with carte de restrictions

Answer 4

-carte génétique= gives order and frequency of restriction sites

• carte de restrictions= gives position relative des sites de restriction dans un ADN

Question 5

different types of ADN vectoriels

Answer 5

1) plasmides (can insert inserts for 10kb and smaller)
- vecteurs de clonage (classique and TA, TA are the vecteurs that have adenosine and thymidine overhangs)
-vecteurs d’expression (ajout d’épitoge and ajout de fluorophores)

• the vecteurs de clonage are used for cloning
• vecteurs d’expression are used for expressing genes or specific proteins
  -adding épitopes = adding short amino acid sequences recognized by antibodies)
• adding fluorophores = adding fluorescent molecules that allows visualisation or tracking or proteins

2) cosmides (can insert inserts bigger than 10kb)

3) phages lambda

Question 6

everything you need to know about plasmides

Answer 6

• circular DNA molecules double brin, different from the DNA chromosomique naturally present in cells
• they maintain a relation de parasitisme or d’endosymbiose with host cell
• duplicated before each cell division (just like chromosomic dna)
• most commonly used plasmides in DNA recombinant technologie are those that replicate in E.coli
• researchers took out undesirable portions naturally present in the plasmide = creates vecteurs plasmidique of around 1,2-3kb

Question 7

plasmides contain 3 main essential regions for dna cloning

Answer 7

1) origine de réplication
2) marquer permettant la sélection
3) region spécialisée pour recevoir un insert d’ADN intérêt (MCS or polylinker)

Question 8

everything you have to know about the 3 essential regions in plasmides

Answer 8

1) origine de réplication
- séquence of 50-100pb
- replication of dna is initiated at this region
- wtv sequence of dna is inserted, is replicated at the same time as the rest of the plasmic dna sequence

2) marquer de sélection
- donne la résistance de la cellule (utilisé pour le sélection des cellules exprimant le plasmide)
-ex: amp, kan, zeo

3) MCS
- contient un polylinker qui contient les séquences de reconnaissances de plusieurs enzymes de restriction
- augmente l’adaptabilité d’un vecteur plasmidique
-chaque site de reconnaissance dans le polylinker est unique = no two sites de reconnaissances dans le polylinker ont la meme sequence d’ADN