Lecture 8 - Recombinant DNA technology : Damian Parry

Question 1

What is the 3 step process for recombinant DNA?

Answer 1

1. Creation of recombinant DNA
2. Cloning/Amplifying the recombinant DNA
3. Expressing/Usage of the recombinant DNA.

Question 2

What are the 4 things you need to create recombinant DNA and why?

Answer 2

Enzymes - to manipulate DNA/RNA.

DNA/RNA - the raw material usually purified from tissue/cell culture.

Vectors - Acts as a vehicle for delivery of the recombinant DNA.

Cells - To amplify and also sometimes site of expression.

Question 3

What types of cells do we use in recombinant DNA technology, and which ones are easiest/harder to use?

Answer 3

Bacterial: The classic choice as they are simple unicellular organisms and are easy to replicate.

Yeast : useful as they are eukaryotic but also unicellular so close to mammals

Mammalian (difficult as they are fragile, have no cell membrane and are complex)

Insect: New! similar to Eukaryotas than yeast.

Question 4

What are the 4 different kinds of enzymes that we need and why?

Answer 4

1. Restriction Enzymes
2. DNA Ligase
3. Taq polymerase (required for PCR to replicate DNA fragment)
4. Reverse Transcriptase (RNA->DNA)

Question 5

What is the function of restriction enzymes?

Answer 5

Recognizes 4-8 base pair palindromic sequences.

Naturally produced by bacteria for self defense against bacteriophages.

Cleaves DNA at specific sequences by binding to x2 strand DNA and then binds tightly to a specific sequence and cleaves the DNA.

Question 6

What are the 2 types of cleavage?

Answer 6

Symmetrical : (blunt ends)

Assymetrical : (sticky ends) with either 3’ or 5’ overhang

Both require breakage of phosphodiester bonds and H Bonds.

Question 7

What is the function of DNA ligase?

Answer 7

To reform phosphodiester bonds and strengthen the new piece of recombinant DNA. Acts as a glue/sealant - it will ligate 2 strands and then detach.

Question 8

What are the desired properties for Vector DNA?

Answer 8

They should contain..

1. Unique restriction sites for insertion of new DNA.
2. An efficient origin of replication.
3. Contain a gene that allows selection of cells which contain the plasmid (e.g antibiotic resistance.)
4. Contain regulatory segment to allow expression of the gene.
5. Must be sturdy and easy to introduce without rejection.

Question 9

What is the usual size of a plasmid in kbp?

Answer 9

2-200kbp.

Question 10

Why are plasmids often used as vectors?

Answer 10

Circular DNA is robust, and plasmids replicate independently of the chromosome.

Question 11

What is a common example of a plasmid?

Answer 11

PUC18

Question 12

What is a polylinker?

Answer 12

A very small section within a plasmid where one can find the restriction sites.

Question 13

What are Cosmids/Phagemids?

Answer 13

Cosmids/Phagemids: : Genetically engineered hybrids that replicate as a plasmid but can be packaged as a bacteriophage (contains cos sequence). They can carry much larger pieces of DNA - but also means they are difficult to manipulate and use.

Question 14

What are the 2 ways in which the “insert” is isolated to produce random non-specific DNA?

Answer 14

Random insert: DNA is isolated from cells and then cleaved with restriction endonuclease.

mRNA alternative: isolate mRNA, and use transcriptase to produce cDNA. mRNA tells you which genes are active - a useful feature!

Question 15

What method do we use to isolate “insert” that will produce specific fragments of DNA?

Answer 15

PCR

Question 16

How do we transfer recombinant DNA into a host/vector? (in this case bacteria)

Answer 16

Add plasmid, mix with baterial cells - to make the cells competent we heat shock them and use CaCl, then incubate at 37 C for 30 mins then spread on plate.

Question 17

What is competence and why does it need to be induced?

Answer 17

Makes cells porous, and therefore more likely to take up the recombinant Plasmid. Bacteria are not naturally competent, which is a defense mechanism to fight back against things such as bacteriophages.

Question 18

What is insertional inactivation?

Answer 18

A way to recognize which plasmids have the insert.

E.g in PUC18, insertion of dna into polylinker disrupts lacZ gene, so inactive beta galactosidase means the bacteria are colourless.

Blue = useless
Colourless = desired

Question 19

What are 3 other selection tests?

Answer 19

1. Hybridisation of ssDNA probe complementary to sequence of interest.
2. PCR using primers specific to site of interest.
3. Screen for expression of protein encoded by site of interest (e.g enzyme activity or reaction with antibody)