Restriction Enzymes & Molecular Cloning

Question 1

techniques for analysing DNA

Answer 1

• Cleavage of DNA , specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual pieces of a genome
• DNA ligation, which makes it possible to seamlessly join together DNA molecules from widely different sources
• DNA ligation a portion of a genome (often an individual gene) is “purified” away from the remainder of the genome by repeatedly copying it to generate many billions of identical molecules

Question 2

how are restricted enzymes obtained and what do they do ?

Answer 2

• purified from bacteria
• Cut DNA double hlix at specific sites defined by the local nucleotide sequuence
• Cleave a long double stranded DNA molecule into fragments of strictly defined sizes

Question 3

how are restricted enzymes obtained and what do they do ?

Answer 3

• purified from bacteria
• Cut DNA double hlix at specific sites defined by the local nucleotide sequuence
• Cleave a long double stranded DNA molecule into fragments of strictly defined sizes

Question 4

why are enzymes called restriction nucleases ?

Answer 4

because these enzymes restrict the transfer of DNA into the bacteria

Question 5

are long are target sequences typically ?

Answer 5

4 to 8 nucleotide pairs

Question 6

what does restriction nuleases do ?

Answer 6

• They cut a particular DNA molecule at the same sites reliability generating the same sat of DNA fragments

Question 7

Analysis of Restriction Enzyme Digests

Answer 7

• the DNA fragments are loaded unto a gel (e.g. Agrose)
• A voltage is applied

Question 8

Analysis of Restriction Enzyme Digests

Answer 8

• the DNA fragments are loaded unto a gel (e.g. Agrose)
• A voltage is applied

Question 9

is what 2 senses is DNA cloning used ?

Answer 9

• Act of making many identical copies (typically billions) of a DNA molecule—the amplification of a particular DNA sequence
• the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome

Question 10

what are the most commonly used baterial cloning vectors ?

Answer 10

Plasmids

Question 11

How are plasmids introduced into a bacteria ?

Answer 11

By a process called **transfomation **

Question 12

whta type of marker are usually used in plasmids ?

Answer 12

Antibiotic resistance genes
- only cells containing this will survive

Question 13

Applications of Bacterial Gene Expression & Regulation

Answer 13

Blue White Screening

Question 14

what are the 7 steps to clone any DNA fragment ?

Answer 14

1. Choice of host organism and cloning vector
2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Creation of recombinant DNA
5. Introduction of recombinant DNA into host organism
6. Selection of organisms containing recombinant DNA
7. Screening for clones with desired DNA inserts and biological properties

Question 14

what are the 7 steps to clone any DNA fragment ?

Answer 14

1. Choice of host organism and cloning vector
2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Creation of recombinant DNA
5. Introduction of recombinant DNA into host organism
6. Selection of organisms containing recombinant DNA
7. Screening for clones with desired DNA inserts and biological properties

Question 15

Recombinant Insulin Production

Answer 15

1. insert gens for each of the two insulin polypeptides next to a highly expressed genes for beta-galactosidase
2. Tranform E.coli with the recombinant expression vectors, and select transgenic cells by their antibiotic resistance
3. Purify fusion prioteins
4. Chemically remove insulin polypepides from the beta-galactosidase protein
5. combine the polypeptides to produce functional insulin
   6.