Flashcards in Biochemistry Lecture 3 - DNA Techniques Deck (18):
How is bacterial DNA protected from its own endonucleases?
Bacterial DNA is methylated (by methylase), so the endonucleases are unable to cleave the bacterial DNA, only infecting phage DNA that is unmethylated.
Are restriction endonuclease sites palindromic?
What pole does DNA migrate to during electrophoresis and why?
DNA migrates to the (+) anode because DNA is negatively charged.
What is pulse field gel electrophoresis used for?
To analyze very large DNA fragments, even chromosomes.
What does Southern Blot analysis help you find out?
One can find the size and gene locations by adding more restriction endonucleases and creating a restriction map. You can also look to see if certain genes underwent a deletion or insertion mutation.
How could you use Southern Blot to diagnose Sickle Cell Anemia?
The beta-globin gene undergoes a mutation from GAG to GTG, so you would use a restriction endonuclease designed to cleave the wild type gene. If electrophoresis shows two fragments (two alleles), then that indicates the wild type allele was cleaved and the mutated allele was not.
What does Northern Blot analyze?
How do you analyze different people's RFLP's on a Southern Blot?
Obtain a fluorescent probe that will hybridize to a gene on both samples, and cut the samples with the same endonuclease(s). The probed fragments will migrate differently because the two DNA samples would be cleaved differently. Some RFLPs correlate with disease (know this).
What is FISH, how does it work, and what does it test for?
Fluorescent In-Situ Hybridization. Chromosomes are spread out on a slide and carefully denatured so that the chromosomes are still recognizable. Then a fluorescent probe is applied that will bind to cDNA sites. This checks for the presence, absence, or copy number of chromosomes.
What is the purpose of SDS in a Western Blot?
SDS denatures proteins and gives them the same negative charge so that they can be separated by size alone. A radiolabled antibody is then applied to locate a specific protein on the polymer sheet.
What does the DNA Band Shift technique test for?
It tests for DNA-protein interaction. It is essentially a Southern Blot except you add a protein to see if the protein will bind to the DNA, retarding the DNA-protein fragment during electrophoresis.
What does a DNase I footprinting assay tell you?
It gives you specific into about where a protein-DNA interaction occurs. A DNA strand is asymmetrically radiolabeled and digested with DNase. Another identical strand is labeled and mixed with a protein, then digested. The bound protein will prevent digestion, so when the two mixtures are ran on a gel, there will be a big footprint where the digestion did not occur. This is where the protein was bound.
Why aren't the polymerases used in PCR as processive as endogenous polymerases?
We haven't engineered a PCNA-like molecule to add to PCR reactions, so the PCR polymerases fall off after a certain number of base pairs.
What does Single-stranded Conformation Polymorphism (SSCP) test for?
It tests for mutations. It involves running the suspected mutated PCR product and a wild-type PCR product in a fancy gel. The mutation likely induces a conformational change in the fragment and they will run differently on the gel.
Know the basics of gene cloning, ok?
What is a DNA library?
A shitload of DNA fragments (that comprise of all the DNA you wanna save) that are inserted into vectors and grown in bacteria.
What purpose do ddNTPs serve in sequencing?
They are chain terminators, and if four separate reaction tubes contain different ddNTPs and differently colored labels, you can run a gel, transfer to membrane and read off the sequence based on color and size.