Recombinant DNA Tech

Question 1

Describe the steps in generating recombinant DNA?

Answer 1

Isolate the DNA you wish to clone/copy

Using restriction enzymes fragment the DNA so that it has sticky or blunt ends. (Restriction enzymes are natural found in bacteria)

Insert the DNA fragments into the chosen cloning vector with DNA ligase

Add the recombinant into the new host and induce expression of the gene.

Question 2

What are the vectors used for the creation of recombinant DNA?

Answer 2

Plasmids - small: <20kb

Phages/viruses - medium 9-25kb

Cosmids - medium/large: 30-457kb

Artificial chromosomes - large: 100-1000kb

• yeast
• bacteria

Question 3

What are the clinical applications of PCR?

Answer 3

• generation of DNA fragments for sequencing
• amplification of genes with culturing microbes
• diagnosis of condition: AIDS, Lyme Disease, chlamydia, tuberculosis, hepatitis or HPV
• gene cloning

Question 4

What’s the difference between a genomic DNA library and cDNA library?

Answer 4

Genomic is the entire rganisms DNA coding while a cDNA library represents only expressed genes.

Question 5

What are the common host cells? What features are removed and how is DNA added?

Answer 5

E. Coli and S.cerevisiae

Host cells lack restriction enzymes and recA
-so that they cant destroy the DNA you insert or change it.

DNA is added through transformation (chemical/heat) or electroporation (voltage)

Question 6

What the method of isolating and purification of the desired protien?

Answer 6

Through polyhistidine tagging (His-tagging) which is isolated after the process by metal ions Ni+ or Co+

Must be tagged before inserted into host. Culture. Lyse. Apply the metal. Wash with buffer, important for removing unwanted material. Release the his-tag using imidazaole (out-compete)

Gel electrophoresis

Question 7

Characteristics of plasmids

Answer 7

Small
Autonomous replication 
-origin of rep
-selectable marker
-mmulticloninig site or polylinker: where the gene is placed 

Evaluated by blue-white screen: white is the colonies with the desired gene

Question 8

Cosmids

Answer 8

Have both phage and plasmid characteristics

Selectivity filter, multiple cloning sites from plasmids and a cos site from a gamma phage
The phage inserts the recombinant dna into E. Coli

For larger genes than plasmids.

Do not exist in nature

Question 9

Artificial chromosomes

Answer 9

For cloning of very large genes

Can be either bacterial or yeast
Yeast are eukaryotic so DNA with introns and post-translational modifications can be performed by yeast. Bacteria cannot undergo these functions