Lecture 12 Notes

Question 1

recombinant DNA

Answer 1

• is regulated
• NIH Office of Biotechnology activites
• Institutional Biosafety committees

Question 2

restriction enzymes

Answer 2

• bacteria evolved restriction/modification systems to protect their own DNA from alteration
• cut foreign DNA, while modifying their own DNA to protect it
• two components to the restriction system: restriction enzyme & methylase
• site specific endonucleases
• blunt ends, sticky ends

Question 3

gel electrophoresis

Answer 3

• separates DNA fragments according to size
• gels are molecular sieves with different pore sizes
• different types of gels allow separation of different sizes of DNA fragments

Question 4

molecular cloning of fragments of DNA

Answer 4

• a way to isolate specific DNA fragment
• the fragment is purified away all of the other fragments
• the fragment is amplified to create many copies for analysis

Question 5

steps in molecular cloning of DNA

Answer 5

• cut DNA with a restriction enzyme
• ligate DNA to a cloning vector, using DNA ligase
• introduce the ligated DNA into a host bacterium
• detect the desired recombinant clone

Question 6

reasons for DNA cloning

Answer 6

1. isolate an individual gene
   - to characterize it
   - to express the gene product
   - to mutate it in defined ways
2. to isolate all of the DNA/genes of an organism
   - for DNA sequencing
   - to create a collection of individual genes/DNA fragemnts

Question 7

genomic library

Answer 7

a large number of recombinant plasmids that collectively contain the entire genome of an organism

Question 8

how to find a gene of interest in the library

Answer 8

1. pre-genomic era:
   - hybridization and related strategies
   - goal is to find one gene
2. genomic era
   - DNA sequencing
   - goal is to identify all clones in the library

Question 9

dideoxynucleotides

Answer 9

• key to enzymatic sequencing by synthesis
• inhibit chain elongation
• used in ESanger method
• DNA to be sequenced is used as a template for synthesis in the presence f ddNTPs
• a nested set of synthesis products is produced in each reaction due to the presence of ddNTPs