Exam 3 - Lecture 18 Flashcards
what is genetic engineering?
deliberate modification of an organism’s genetic information by directly changing the sequence of nucleic acids in its genome
what is recombinant DNA?
artificially created DNA sequences resulting from combining 2 strands of DNA together
what is cloning?
generating a large number of genetically identical DNA molecules
what is biotechnology?
use of biological organisms to form useful products
what is industrial microbiology?
use of microbes to manufacture important compounds
what are two main reasons you might want to express a foreign gene in a host cell?
- to determine its function
- to purify it
what are four recombinant DNA technologies?
- restriction enzymes
- genetic cloning
- PCR
- DNA sequencing
what do restriction enzymes (RE) do?
they recognize and bind specific sequences in DNA called recognition sites to cleave DNA
where do the following RE cleave DNA?
- Type I
- Type II
- Type III
- Type I: cleave at a defined distance from the recognition site
- Type II: cleave at/around the recognition site
- Type III: cleave at a defined distance from the recognition site
cleavage may cause what kinds of ends in the DNA target?
- blunt ends
- sticky ends
true or false: many restriction enzyme sites are palindromes.
true
what is a vector?
a carrier of foreign DNA
what are four types of cloning vectors? which is the most common*?
- phages and viruses
- cosmids
- artificial chromosomes
- plasmids*
to clone a gene, __________ _____ is combined with/inserted into a cloning vector.
foreign DNA
what are two characteristics of good cloning vectors?
- easy to purify
- replicate autonomously
what are three requirements for vectors?
- origin of replication
- selectable marker (antibiotic resistance gene)
- multiple cloning site (MCS)
what is an MCS and what is it used for?
- multiple cloning site
- site where cloned gene is inserted into the plasmid vector
- contains many unique RE sites
- may contain selection gene (ex: lacZ)
what is the most common bacterial host for insertion of recombinant DNA?
E. coli
what is the most common eukaryotic host for insertion of recombinant DNA?
S. cerevisiae
what are two mechanisms for artificially induced competence for transformation?
- CaCl2
- electroporation
what is PCR?
- polymerase chain reaction
- technique that enables DNA amplification
- rapid synthesis of many copies of a specific DNA fragment
- individual DNA fragments can be specifically amplified from a complex mixture of DNA
what determines the specificity of PCR?
- DNA primers (oligonucleotides)
what is the PCR cycle?
- DNA is denatured
- primers anneal to target DNA
- target DNA is synthesized (amplified)
- repeat x 34 times
PCR causes _____________ apmlification.
exponential
what are the uses of PCR?
- simplifies gene coding
- generates DNA fragments for nucleotide sequencing
- may amplify environmental genes without culturing the microbes
- diagnostic purposes
what is the most commonly used method for determining DNA sequences?
Sanger DNA sequencing
what type of nucleotides does the Sanger DNA sequencing method use to signal termination?
dideoxynucleoside triphosphates (ddNTPs)
strand synthesis terminates when a ________ is incorporated in Sanger DNA sequencing.
ddNTP
