Flashcards in Lecture 27-Analytical Tools in Molecular Biology Deck (40):
Restriction enzymes are named based on _____.
- what bacteria they're expressed in
What two ways can restriction enzymes cut and what do each result in?
- asymmetrically: 5' sticky ends
- symmetrically: blunt ends. Usually from cutting in palindromes
Restriction Modification System
- bacterial system that protects host from restriction enzymes via methylation of the recognition sequence by DNA methylase
Type I restriction enzyme (3)
- cleaves ~1000bp from recognition sequence
- endonuclease and methylase activities
- requires ATP
Type II restriction enzyme (3)
- cleaves very specific sequences by recognizing palindromes
- does NOT require ATP
- used in research because of their specificity
Type III restriction enzyme (3)
- cleaves ~25bp from recognition sequence
- endonuclease and methylase activities
- uses ATP
restriction enzymes allow us to make ______
- first plasmid constructed
- has "unique" restriction sites (only one for each restriction enzyme (Pst1, EcoR1, BamH1, Sal1, PvuII
What kinds of ends does EcoR1 make?
In order to insert a chromosomal segment into a plasmid you have to isolate both segments using _______
- the same restriction enzyme
What happens if there is not the restriction site you need on the plasmid?
- use a polysynthetic linker which contains many restriction sites and ligate it into the plasmid cloning vector with EcoR1 sites
NOTE: not 100% efficient because each step is not 100% efficient
- cleave cloning vector and chromosomal DNA with same restriction endonuclease
- ligate chromosomal DNA into vector to make the recombinant vector
- recombinant vector inserted into cell by keeping cell at 0º with CaCl2, then heat shocking it to open up the cell wall
- cell divides with varying amounts of plasmid (since it replicates independently of the host genome)
Why would you put a restriction site into an antibiotic resistance gene? Give an example using pBR322 and amp/tet resistance of why.
- a way to select for those bacteria that have the gene
- pBR322 is cleaved in the middle of the amp resistance gene by Pst1**
- foreign DNA is ligated into the plasmid disrupting the amp resistance, tet resistance remains intact because its in another spot on the plasmid
- cells transformed and grown on agar with tet for selection
- colonies that grow have plasmids so each colony is divided in half and put on either a tet only control agar plate or a tet + amp agar plate that will kill cells that DO have the gene of interest. Note that the colonies have to be placed in order to know which colony corresponds to which
- those that dont grow on the amp + tet plate but DO grow on the tet only plate have both the plasmid and the gene of interest
Why use bacteriophages instead of plasmids?
- contain more DNA (~60kb) so you can insert 20-40K bp vs. 200-2000 in plasmids
How do you use bacteriophage transformation?
- restriction nucleases cut out the center "filler" DNA
- foreign DNA fragments, also cut by the same restriction endonuclease mixed in and presumably the ends of the original bacteriophage genome attaches to new DNA. This is required because these parts are needed for viral packaging. If either of the ends are missing or if the filler segment is too small it won't go into the bacteriophage
- DNA is packaged into lambda bacteriophages
What is the basic difference between a plasmid and a BAC plasmid? (2)
- BACs have par gene
- BACs have chloramphenicol resistance
- BACs contain multiple restriction sites (or cloning sites) for multiple restriction enzymes in lacZ gene
Why use a BAC over a plasmid or bacteriophage?
- can insert larger pieces of DNA and can be used to look for recombination events in the DNA-donors DNA.
Par gene. Why is this important?
- allows the BAC to only replicate once so there will only be one copy in each bacteria
- since we're going to put a huge piece of DNA in there, if we put more than one there are going to be recombination events. We don't want recombination events because these are used to diagnose recombination events in humans so we don't want it going on in the bacteria as well.
How do you make a BAC containing cell?
- restriction enzyme cuts in the lacZ gene (preventing it from making beta-galactosidase) of the BAC as well as the DNA of interest
- they're annealed together via ligase
- have to use bacteria with a specialized cell wall that will allow plasmids in in response to electric current
- plate bacteria on agar with chloramphenicol (for selection) and a substrate for beta-galactosidase that becomes blue when metabolized. This will tell you whether or not the gene is in the BAC because the lacZ gene should be disrupted by the insert and so the white colonies are the one with the insert
Describe PCR process
- heat DNA to separate strands
- add synthetic oligo primers (~18bp) in excess
- add thermostable Taq polymerase to catalyze 5'-->3' synthesis
- repeat all steps for 25 cycles to get about 1 mill copies
How is PCR different if you're doing it for insertion into a vector for cloning?
- you use primers with non-complementary regions with cleavage site for restriction endonucleases
- after the cycles use endonuclease to cut out sequences
- polysaccharide that's heated into solution to make a gel
What is the difference between RNA gels and DNA gels?
- RNA requires denaturants to prevent formation of 2º structures and keep them in their linear form (formaldehyde for agarose gels, urea for acrylamide gels)
- DNA doesn't make 2º structures so the gels are non-denaturing
What stains DNA/RNA segments so they fluoresce under UV light and how does it work?
- EtBr intercalates between G and Cs
- monomer that forms pores in the gel
- used to separate smaller fragments
What is the difference between agarose and polyacrylamide gels?
- polyacrylamide gels are thinner so they need to stand up and agarose gels do not
What kind of acrylamide gel is used for proteins?
- SDS-PAGE (has SDS detergent that has a slight negative charge so the proteins will be separated only based on size)
After you restriction digest test DNA/RNA why can't you stain with EtBr? How do you find your bad of interest?
- it will make smears
- you put the DNA onto nylon membrane or nitrocellulose paper and probe with radioactively labeled DNA
Why are Southern blots good for quantitative analyses? (3)
- can show change in copy # of a gene
- detects relatively large structural changes of a gene
- detects point mutations if they result in RFLPs
Why are Northern blots good for quantitative analyses? (2)
- can show changes in mRNA amount
- can show changes in RNA size
What must you do to DNA and how before you put it on Watman paper for a southern blot?
- denature it to ssDNA
What is the order of the stack for a southern or northern blot transfer?
Bottom to top:
buffer --> Watman paper --> gel --> nylon paper/nitrocellulose --> paper towels, weight
Why does the stack help transfer DNA onto nitrocellulose paper?
- capillary action from the buffer being drawn up and pulling the DNA with it onto the nitrocellulose paper
How do you find your bands of interest on a northern or southern blot that has already been transferred?
- radioactively labeled probe put in a bag with the paper, probe is in excess
Why does everyone's DNA fragment differently in response to restriction enzymes?
- while our coding and regulatory regions are under evolutionary pressure and must therefore remain the same, the other 20% of our DNA is not and this may have allowed them to develop restriction sites in different places that can vary from person to person.
In forensics, the DNA probes target _______ on southern blots.
What is the advantage to microarrays? Ex?
- can look at many genes at the same time to determine relative expression
- oocytes vs. tadpole to determine gene expression at different stages of development)
- normal vs. malignant cells
How does a microarray work?
- different DNA sequences are located on precise spots on a glass slide and each spot corresponds to a different gene
- isolate complete compliment mRNAs from 2 different specimens
- use RT to convert them to cDNAs
- fluorescently probe each set of specimens with red or green
- expose both specimens to the microarray chip at same time and allow them to anneal
- spots that fluoresce with one higher than the other have higher levels of expression of that gene and vice versa for the other color. Yellow spots have equal amounts of both.
Describe Sanger dideoxy sequencing
- 4 reactions each w/3 normal nucleotides and 1 type of nucleotide population thats part dNTP and part ddNTP
- when ddNTP is incorporated during strand synthesis you terminate elongation of the strand
- radioactivity is in the primer or added during synthesis reaction
- each reaction is run in different lanes of a gel
- gel is read from bottom (smallest sequences) to top (largest sequences)