Gene Isolation &Manipulation Flashcards
What protects the host genome from being digested?
methylation of A or C
Restriction enzymes…
…digest DNA at specific sequences
…many are palindromes
Are larger restriction sites recognized more often or less often?
less often
What is more efficient in terms of ligating together? Blunt ends or sticky ends
-Sticky ends are more efficient
What does DNA ligase do?
- seals DNA molecules that have been ligated together
- forms phosphodiester bonds
- produces a covalent circular recombinant molecule
A circular DNA with one restriction enzyme site is cut with a restriction enzyme. How many half sites does it produce?
- 2
- if 2 half sites are ligated together, the restriction enzyme has 2 sites
What is in a ligation mix?
non-ligated linear DNA and ligated circular DNA
-the reaction is not 100% efficient so it contains both elements
Explain how cells are transformed with DNA.
- ligation mix is added to competent E.coli. cells that have been treated with CaCl2. CaCl2 makes holes in the bacterial cell wall which inreases the rate of DNA uptake
- ligation mix enters the E.coli. cells and is replicated
(the circular plasmid will replicate because they contain an ORI, non-ligated linear molecules do not replicate)
What is a copy number?
the number of plasmids per cell
- can vary from 1-700
- the most reliable ones are about 10 kb
Are plasmids that contain pMB1origin of replication integrated into BAC?
No
-have a high copy number
pUC18
-has multiple cloning sites that are next to each other and are unique
Explain how to get a blue or white colony using a blue/white screen.
- Blue: beta-gal cleaves the substrate X-gal
- White: DNA is cloned into a multiple cloning site so beta-gal cannot be made because the lacA-alpha gene has been disrupted
Explain how a blue/white screen works.
- allows for selection of recombinant plasmids
- lactose is cleaved into its monosaccharides by beta-gal
- beta-gal has 2 subunits: lacZ alpha and lacZ omega
- lacZ alpha is deleted from the host genome
What are YACs and BACs used for?
- cloning large pieces of DNA
- YAC: 200kb
- BAC: 100kb, ORF comes from F episome
- F episome has a copy number of 1 (low)
Describe the process of southern blotting.
- DNA is separated by gel electrophoresis
- transferred to a membrane as ssDNA
- incubated with a probe. Probe H-bonds using complementary bp
- nitrocellulose paper can be placed to be visualized by X-ray film (autoradiography)
Describe northern blotting.
- RNA is placed on nitrocellulose paper , immobilizing it
- then hybridized to radioactive DNA or RNA probes
- determines time or tissue where gene is transcribed into mRNA
How is In situ RNA hybridization done?
- can be done with digoxygenin labeled antisense RNA that hybridizes to sense mRNA
- Dig-UTP is incorporated into antisense probe
- Dig is detected by an antibody
Describe the process of Western Immunoblot.
- protein is separated by SDS-PAGE
- transferred to nitrocellulose paper
- Nitrocellulose is incubated with a primary antibody that recognizes the protein
- then incubated w/ a secondary antibody that recognizes the primary antibody and has an enzyme attached to it
- when substrate of the enzyme is converted, the location of the primary antibody can be observed
Describe how dideoxy sanger DNA sequencing is done.
Dideoxy nucelotides are incorporated into a DNA strand and act as chain terminators because they do not have a 3’ OH.
- there are fluorescent labels for each ddNTP
- each reaction has regular dNTPs so that DNA synthesis is not immediately terminated
- when a ddNTP is added, DNA synthesis is terminated
- end up w/ a pool of extended mcs
- mcs are separated by electrophoresis
- fluorecent mcs go past detection window, emit fluorescence that is recorded on a chromatogram
What is PCR?
- start with a template DNA and specific primers
- priers hybridize to each end of the gene
- need 2 primers (1 for each strand) & excess dNTPs
What are the steps for a PCR reaction?
- Denature template DNA and primers at 95 C
- Anneal primers to ss template DNA at 55 C
- Taq polymerase extends primers using excess dNTPs at 72 C
- repeats 30 times
- amplified DNA increases exponentially
PCR benefits
- primers can be designed if a sequence is known from cDNA or genomic DNA
- cDNA is made by reverse transcriptase
- PCR products that are amplified by cDNA can reveal how a gene is spliced
- can determine RNA level
Describe real-time qPCR
- used instead of northern blots
- RNA is copied into cDNA with reverse transcriptase
- cDNA pool will reflect amount of mRNAs
- cDNA pool is used as a template for PCR
- SYBR green is added to PCR reaction (fluoresces)
- fluorescence is measured in real time to track PCR progress
- more abundant mRNA starts amplifying sooner
- less abundant mRNA amplifies after more cycles
Describe high throughput DNA sequencing.
-Solexa coupled with Illumina
-get diff. fragments with known sequences at each end
-cluster generation by bridge amplification in the flow cell
-sequence by synthesis
-Illumia depends on a reversible terminator, FL-nucleotide acts as a chain terminator
-2nd round of elongation
-fluorescent tag is removed
-Illumina reads 75-100 bp at a time but can do many reaction at once
-
