cloning

Question 1

Uses of recombinant DNA technology

Answer 1

• production of therapeutic proteins
• investigation of function of genes, enhancer elements, etc.
• generation of novel proteins
• generation of recombinant vectors for gene therapy
• making transgenic plants and animals

Question 2

How do restriction endonucleases form part of bacterial defense systems

Answer 2

Restriction endonuclease binds to recognition site on invading virus and cleavages both of the DNA strands

• own DNA not targeted as the recognition sites are methylated and bind to methyl transferase

Question 3

What are the types of restriction enzymes

Answer 3

1. Isoschizomers

2. Neoschizomes

Question 4

What are isoshizomers

Answer 4

Restriction enzymes that recognize the same sequence and cut it in the same way

Question 5

What are neoschizomers

Answer 5

Restriction enzymes that recognize the same sequence and cute it in different ways (to produce different sticky ends)

Question 6

Components of plasmid vectors

Answer 6

• circular
• origin of replication
• antibiotic resistance gene + lacZ gene
• MCS

Question 7

Components of expression vectors

Answer 7

• origin of replication
• antibiotic resistance gene (AmpR)
• T7 promoter
• RBS- ribosome binding site
• T7 terminator
• BamHI site for inserting CDS

Question 8

Why don’t expression vectors have MCS like plasmid vectors

Answer 8

Because for coding sequence to be read correctly in frame needs to be inserted in specific region

Question 9

Characteristics of cloning bacteria

Answer 9

• specialized strains of E. Coli
• lack methylation enzymes (so restriction sites aren’t silenced)
• lack recombination ability
• sensitive to specific antibiotics
• partial laxZ in genome

Question 10

How does directional cloning occur

Answer 10

2 different enzymes produce 2 non compatible ends of the plasmid

Question 11

What is Sanger sequencing

Answer 11

A method to determine the base nucleotide sequence in DNA

- en corporation of ddNTP which lacks OH group results in no further elongation once included

Question 12

Sequence of PCR (polymerase chain reaction)

Answer 12

1. DNA heated to 90-100 degrees to separate the 2 strands
2. The DNA quickly cooled to 30-65 degrees to allow short single stranded primers to anneal their complementary sequences
3. Solution heated to 60-70 degrees, DNA polymerase synthesizes new DNA strands, creating 2 new double stranded dna molecules

Question 13

What type of dna is used in PCR

Answer 13

Archaea dna used as it can withstand without denaturing like E. coli.

Disadvantage is it doesn’t have proofreading properties and so other bacterial enzymes are used in combination to proofread

Question 14

Steps of RT-PCR (reverse transcription PCR)

Answer 14

1. RNA gets converted into DNA by reverse transcriptase and DNA polymerase
2. RNA gets degraded by RNase
3. Normal PCR of DNA