Tools of Molecular Biology Flashcards
What is autoradiography
Another way to visualize DNA bands. You transfer bands to an xray film and then expose the film to radiation.
Describe stringency and the factors that affect stringency.
Higher stringency requires a more perfect match between probe and DNA as compared to low stringency.
Temperature: high stringency = high
Salt concentration: higher salt concentration, lower stringency
Salt stabilizes the hybridization (lowers melting point)
Amount of denaturant, more denaturant, higher stringency
Probe length - shorter probe increases stringency, longer probe decreases stringency.
Restriction Fragment Length Polymorphism
A way to track the appearance and disappearance of restriction enzyme sites. INherited differences can be used to identify people. Use PCR and then southern blotting,
Describe the three blots along with microarrays.
Southern blotting, you have fragments of dsDNA, when the buffer is sucked up to transfer onto the nitrocellulose paper. It will denature the DNA and become single stranded on the paper, then you can probe for specific fragments
Northern blotting - measure gene expression. Probe is usually single strand DNA. And good for measuring the expression of one gene but not multiple genes.
Two mRNA samples; you convert all sample to cDNA along with a fluorescent label. Then you allow all of it to hybridize to the single stranded genes of the microarray. Colors will tell you their expression relative to each other.
Describe ASOs and Allele specific PCR.
The probes match the nucleotide sequence flanking the substitution. Can be used to detect single base mutations. You are using a short probe and under high stringency conditions. You can use the probe to detect the presence of a normal allele or mutated allele. Can be used to detect skicle cell anemia.
ASPCR is based on perfect matching of the 3’ end of the primer. Similar to high stringency conditions. If the primer perfectly binds, only that DNA will be amplified.
Reverse transcriptase PCR
Good for quantifying gene expression. You take the RNA, you make the cDNA, remove the RNA and only amplify the cDNA. Its good for evaluating real time changes in gene expression.
Describe sanger method and automated DNA sequencing.
Sanger: four tubes, each contains a different kind of dideoxynucleotide. You insert the target DNA, add primers, polymerase and normal dNTPs. It will start to replicate the strand in each tube but stop with the incorporation of each nucleotide. Each nucleotide has a different signal so you can detect. Run a gel, read bottom up.
Automated: is a single tube with all four ddNTPs and the machine detects the incorporation, dunno.
What is the genomic library? What is cDNA library? What are the differences?
Genomic library is the total DNA in an organism cut up into fragments by restriction enzymes and ligated into plasmids and transformed into bacteria. Each line of bacteria contains a fragment of the library. If you want to isolate a DNA fragment/gene you can use the probe to isolate the colony carrying the DNA clone
CDNA library is taking mRNA, making it into cDNA, inserting them into plasmids and then cloning. If you want to locate a gene, you can hybridize and see if the gene is being expressed. Not only that, you can measure level of expression. You take a probe and isolate the clones that express a gene. You can see different levels of expression.
What is serial DNA cloning:
inserting a set a DNA fragments from different sources through sequential cuts, additions, cuts and additions. For example GFP. We add the gene. Then we add the GFP. If that gene is expressed we can monitor it through the fluorescence. then we can localize where it is expressed, and when it is expressed.
Epitope tagging
Good for isolating purified protein from a recombinant. We use an expression vector with a strong promoter and inserted is also an epitope for an antibody. Extract is run through a column full of antibodies and we can isolate the pure protein.
What are the three ways animals can be genetically altered to observe gene function?
Gene replacement: we replace the normal gene with a defective one. See the effects of the mutant without interference of the normal
Gene knockdown: remove or knockdown expression of the gene to see its function
Gene addition: add the mutant gene with the normal gene. Look at how one gene may overpower the other one etc.
