Tools in RDT

Question 1

List the requirements for molecular cloning? And the basic steps? Tools? Techniques?

Answer 1

foreign dna

host org.

vector

means to insert

obtaining enough

selecting & screening

Question 2

what are the products of ligation reaction?

Answer 2

2F +V , V + F, 2V + 2F, F + 2V

Question 3

Why MOs are used here?

Answer 3

growth req.s are minimum

short gen. time

less space needed

haploid

low cost & easy

Question 4

Why E.Coli is used?

Answer 4

grows rapidly

non expensive

easy to work with

easy to transform

some are non-patho

Question 5

Mutation rate equation? Scope of mutation?

Answer 5

of mutations/total # of cell gen.s

Question 6

Explain each type of mutants

Answer 6

auxotrophs

resistant mutants

conditional lethal mutants

Question 7

List the methods of gene transfer b/w bacteria

Answer 7

transduction

transformation

conjugation

Question 8

What is transformation?

Answer 8

genetic after free dna incorporation

Question 9

What is competence? explain.

Answer 9

cells that can take up dna

strains

inherited

artificially induced

specific proteins control

• mem. assoc. dna binding protein
• nucleases

Question 10

What are the 2 competence induction methods? explain

Answer 10

chem - high ca2+

phys - electrophoration

• high V
• transient pores formed — dna uptake thru

Question 11

integration of transforming dna? steps? recombinase?

Answer 11

bind to the surface

assoc. with competence specific protein

remain attached to the dna recombinant process – recombinase

Question 12

What is transduction?

Answer 12

dna transformed to cell to cell thru viruses

Question 13

what are the phases of lytic cycle?

Answer 13

attachment
penetration
biosynthesis
maturation
lysis

Question 14

Visualize the process of transduction

Answer 14

ss

Question 15

What are the 2 types of transduction?

Answer 15

general - transfer dna from one bac to another - low f

special - extreme efficient transfer while allowing a small region of bac. chromo. to be replicated indie of the rest

Question 16

What is conjugation? Describe the steps in transformation.

Answer 16

cell to cell contact and a conjugate plasmid in the donor cell?

Question 17

What are Hfr cells? How it conjugates with a F- cell?

Answer 17

high f - F+ plasmid integrate into the chromosome

Question 18

Explain the rolling circle replication process?

Answer 18

ds circ. dna - nick - 5’ - roll

Question 19

What is transfection? explain the process.

Answer 19

injection of dna

dna + liposome – lipoplex – endocytosis – endosome

• -lipid mixing – escape to nucleus
• -maturation – dna fragmentation in lysosome