03/05/2012 Genetics CQ2: In a northern blot of total RNA staining the blot before you transfer tell you

(e) all of these 1. Probing the blot a. Uses hybridization to labeled probe bind to sequence of interest b. Size and intensity matters c. Information i. Stain on top ii. Used three different probes (enzymes being studied) iii. See different transcripts depending on size of bands iv. See different levels v. Size of the transcript processed; alt splicing vi. Gene expression 2. Hybridization: effect on Stringency on blots a. High stringency: not a lot binds (see one band) i. High Temp; High stringency ii. Low Salt; High stringency b. Low stringency: multiple bands i. Base pairing b/t strands with few differences ii. Low Temp; High stringency iii. High Salt; High stringency c. Hybridization depends on it d. Sequence of probe GC content e. % match with target f. Temperature g. Salt concentration i. Charge on Phosphate group repel the base pair ii. Increased salt shields negative charge from in other 1. Not much hydrogen bonding needed to keep helix together CQ.3 (a) need more hydrogen bonds to hold them together 3. How else to look at mRNA levels a. Use Reverse-transcriptase for PCR i. Need to transcribe RNA to cDNA using reverse transcriptase ii. Perform PCR 1. RT-PCR iii. Amount of cDNA should reflect amount of mRNA b. PCR i. 3 Steps

1. Melting 2. Annealing a. specific primers added i. for polymerase 3. Elongation ii. Dif Temps 1. 95 C a. Normal melting temperature 2. Variable 30 60C a. Depends the GC content of primer b. How long the primer is (~20-30bp) 3. 72 C a. Taq Polymerase iii. Repeat 20-30 cycles to make millions of copies CQ 4. The temperature of annealing in PCR depends on (c) the GC content of primer 4. Detecting RNA-RT-PCR gel a. Gel of amplified cDNA i. Gene 1 and Gene 2 b. Stain i. Levels reflect starting RNA 1. 2 different genes a. Two sets of pairs of primers c. T1 cDNA>than C2 d. T2 cDNA>than C@ e. But not for all genes f. As PCR come up some point it levels off i. If look too late might not be changing due to maxing out the amount you can detect ii. Solution 1. Real time PCR or Quantitative PCR 5. Quantitative PCR (Q-PCR) a. Monitor PCR as its proceeding to get rate b. Monitor using a fluorometer inside PCR machine i. Light reflects product c. Two main ways i. Using dye seeing any fluorescently labeled ds (syBR Green) CQ 5. The blue blobs in the Q-PCR animation are (a) DNA polymerase ii. Using a probe/labeled primer specifically for you gene that has F (Fluophor) and Q (quencher) (TAQ-MAN)

1. As DNA is amplified, more Reported probe is degraded so more and more fluorescence is seen iii. Differences 1. Second: probe is degraded a. Dif Fluorphors on different probes 2. Sybergreen is NOT sequence specific unlike second 6. Enzymes to manipulate DNA a. Copy DNA i. Taq b. Cut DNA i. Nucleases ii. Restriction enzymes c. Paste DNA i. Ligase 7. Restriction Enzymes a. Classified based on sequence cut by enzyme 8. Detecting RFLP differences (Restriction fragment length polymorphism) a. Mutations b. Isolate DNAcut w/ restriction enzyme c. Run on geltransfer probe w/ specific gene (Southern blot) d. For crime anaylsis CQ6. Differences in the genome of the people could also be analyzed using PCR and (e)all of these 9.