Module 6: Molecular biotechnology

Question 1

molecular cloning and recombinant DNA

Answer 1

cloning: introduce foreign DNA into his organism via a vector

recomb DNA: obtain new DNA construct

Question 2

molecular cloning steps (3)

Answer 2

1. gene of interest+ vector=recombinant DNA
2. Recombinate DNA+host
3. produce protein of interest

Question 3

restriction enzyme

Answer 3

enzyme inserting or deleting genetic elements (remove foreign DNA)

Question 4

sticky vs blunt end

Answer 4

sticky: unpaired DNA nucleotide at 5’ or 3’

- more efficient recombination than blunt end

Question 5

transformation, transduction, transfection

Answer 5

introduction of genetic material into host via vector

transformation: plasmid vector
transaction: viral vector
transfection: non viral

Question 6

elements of cloning plasmid

Answer 6

1. origin of replication: initiation of cloning
2. multiple cloning site: insert DNA
3. insert
4. promoter: drives transcription
5. antibiotic resistant gene
6. selectable: allows for bacteria selection

Question 7

virus

Answer 7

• invade host organism to replicate inside cells
• used to deliver genetic material to specific cell types
• virus tropism: specific of virus to target certain cell type

Question 8

expression plasmid

Answer 8

plasmid used to express foreign proteins, on which there is usually a promoter above MCS

Question 9

competent bacteria

Answer 9

bacteria permeable to DNA, to make permeable (create pores):

1. ice bath+heat shock
2. electric field (electroporation)

Question 10

clonal selection

Answer 10

if plasmid contains antibiotic resistance cassette, only bacteria w plasmid form colonies on substrate

Question 11

steps of molecular cloning (5)

Answer 11

1. amplify DNA of interest
2. insert DNA into cloning vector
3. introduce vector into host
4. select organism that incorporate vector
5. confirm protein produced (analysis)

Question 12

PCR (polymerase chain reaction)

Answer 12

1. dna melting at high T (denature DNA)
2. primer annealing
3. addition of polymerase to allow dna replication

Question 13

quantitive per

Answer 13

• amount of dna amplified by PCR doubles each cycle (Theo)

- use formula

Question 14

Nt, Ct, Ni, No

Answer 14

Nt: threshold dna amount
Ct: threshold cycle number
NI; target dna molecules
Ci: cycle to reach Ni

Question 15

gel electrophoresis

Answer 15

separate charged samples based on electrophoretic mobility

• analyst results of molecular cloning
• once cloning vector created, host cells transformed