Lecture 12 Notes Flashcards
1
Q
recombinant DNA
A
- is regulated
- NIH Office of Biotechnology activites
- Institutional Biosafety committees
2
Q
restriction enzymes
A
- 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
3
Q
gel electrophoresis
A
- 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
4
Q
molecular cloning of fragments of DNA
A
- 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
5
Q
steps in molecular cloning of DNA
A
- 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
6
Q
reasons for DNA cloning
A
- isolate an individual gene
- to characterize it
- to express the gene product
- to mutate it in defined ways - to isolate all of the DNA/genes of an organism
- for DNA sequencing
- to create a collection of individual genes/DNA fragemnts
7
Q
genomic library
A
a large number of recombinant plasmids that collectively contain the entire genome of an organism
8
Q
how to find a gene of interest in the library
A
- pre-genomic era:
- hybridization and related strategies
- goal is to find one gene - genomic era
- DNA sequencing
- goal is to identify all clones in the library
9
Q
dideoxynucleotides
A
- 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
