Chapter 20 Flashcards
(22 cards)
Restriction endonucleases
enzymes that cut a DNA molecule at a particular place
DNA ligase
seals nicks in the DNA backbone, covalently bonding the two strands
Vectors
- Carrier DNA molecules
- Carry selectable gene marker to distinguish cells that have taken them up from those that have not
Transformation - purpose
To introduce plasmids into bacteria
Transformation - 2 main techniques
- Heat shock
2. Electroporation (electricity)
BACs
- Bacterial Artificial Chromosomes
- Generally very large but low copy number of plasmids
YACs
- Yeast Artificial Chromosomes
- have telomeres at each end, ORI, and centromere
Expression Vectors
- Designed to ensure mRNA expression of cloned gene
DNA Libraries - 2 types
- Genomic
2. Complementary (cDNA)
Genomic Library
Contains at least one copy of all sequences in genome of interest
cDNA library
contains complementary DNA copies made from mRNAs present in the cell’s population
Reverse transcriptase - function
Generates cDNA from mRNA
What does reverse transcriptase produce?
mRNA-DNA double-stranded hybrid molecule
What do probes do?
sort through library and isolate specific genes of interest
What is a probe?
any DNA or RNA sequence complementary to the target gene of sequence being identified
PCR - 4 requirements
- DNA Polymerase
- DNA Template
- Primers
- Salt (such as magnesium)
PCR - 3 steps
- Heat up to denature
- Primers anneal to the template
- Primer extension
Southern blot - 5 steps
- DNA samples that are cut with restriction enzymes are loaded on agarose gel for electrophoresis
- DNA is separated by electrophoresis
- DNA-binding filter… placed on gel
- Filter placed in heat-sealed bag/tube with solution containing labeled probe
- Bound probe detected by film or probe signal capture with a digital camera
Southern vs Northern vs Western blot
Southern: DNA-DNA hybridization
Northern: RNA-DNA hybridization
Western: Proteins
Agrobacterium-mediated plant transformation
bacterial chromosome is put into Ti plasmid
Particle bombardment plant transformation
- coat DNA in gold
- place into tubing
- cut tubing into 1-2 mm length
- put in chamber, connect to high pressure can, and use pressure to bombard the sample
Using Embryonic Stem cells, scientists introduce targeting vectors into cells via _____.
Electroporation
