BCHM 4116 1st Edition Lecture 9Outline of Last Lecture I. Gibson AssemblyII. Gateway Cloning (Recombination-based cloning)III. DNA library ConstructionIV. Genomic DNA library construction: Phage vector, human DNAV. Making a cDNA libraryOutline of Current Lecture VI. MethodsVII. Southern blotVIII. Factors that determine the stringency of the Southern hybridizationIX. Introduction to MutationsX. Northern blotXI. Library screeningXII. PCRCurrent LectureMethodsHybridization: can be defined as the formation of a double stranded (ds) duplex nucleic acid molecule. When on strand is labeled and used as probe, this technique allows the detection of nucleic acid molecules that are complementary to the known probeBlotting: transferring of DNA/RNA from gel to membrane (filter)In southern blot, DNA are separated by gel electrophoresis, the resulting fragments are transferred (blotted) to a solid support (membrane/filter) and a labeled or tagged probe is used to locate the position of a complementary sequence by hybridizationSimilar hybridization techniques can be used to screen a genomic library for a clone that hybridizes with a specific probe, or to identify a specific RNA (Northern blot) after the separation of a mixture of RNA molecules by electrophoresis. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Southern Blot1. DNA is digested with restriction endonucleases2. Gel electrophoresis is used to separate the fragments by sizea. The bands are visible under UV if soaked in ethidium bromide3. The gel is then soaked in NaOH to denature and then add tris to neutralize4. Nitrocellulose filter (membrane) is used to ‘blot’ the bands onto the paper 5. The DNA fragmetns are bound to the filer paper in positions identical to those on the gel6. This paper is then added to a solution with the probe, which can hybridize with the right DNA strand 7. The filter paper is exposed to x-ray which shows the bands that were able to hybridize with probe How selective you are for the probe to DNA hybridization varies Here is a visualFactors that determine the stringency of the Southern hybridizationThe point is so that the conditions are right so you don’t have a lot of non-specific binding When conditions are right, the probe stays bound, and doesn’t get washed away with non-specific binding instances. Stringency: How close a match between strands is required for duplex formation/maintenance.Relaxed or low stringency conditions permit formation of hybrids with mismatch. High stringency conditions permit only well-matched hybrid formation The stability of the probe-target duplex can be measured by its Tm.Stringency can be adjusted based on reaction conditionsWashing temp < tm you get signalWashing temp > tm you don’t get signal Wash solution: Wash with salt solution (NaCl, sodium acetate), some kind of buffer to keep pH, can also add formamide By changing concentration of salt in washing solution, you can change tm (melting temperature)High concentration of salt in wash, makes Tm higher: less likely to wash away nonspecific bindingLow concentration of salt in wash, makes Tm lower: wash away nonspecific binding easier because its less stable in low salt concentration Formamide: destroys struction, makes it easier to wash away Whatever makes it easier to wash it away, it means that I’ts more picky, high stringency High salt concentration = high tmHigh formamide = lower tm High gc = high tm To calculate Tm for DNA:DNA hybrids:Tm = 81.5°C + 16.6 (log10[Na+]) + 0.41 (% G+C) - 0.63 (% formamide) - 600/N Where N is the length in bp Tm decreases 1-1.5 °C for each 1% mismatch Tm decreases 0.63 °Con avearge for each 1% formamide Tm increases 16 °C for each log increase in cation concentration In practice, the stringency of Southern is often controlled by the washing conditions, most importantly the washing temperature, salt concentration, and formamide. Probes for Southern hybridizationInformation for probe design1. Degenerate oligonucleotides2. Heterologous probes3. Fragment of gene of interestLabeling of the probe1. Random priming and PCR are two of the many methodsNorthern blotThis is the same, except its done on RNA, not DNAGene expression analyzed by Northern blots •RNA samples undergo electrophoresis •RNA separated by molecular weight •Transferred to membrane •Probe labeled  Radioactivity or antibody ligand •Hybridized to RNA on membrane •Hybridization dependent on time, temperature, salt concentration, and nucleic acid sequence and concentration Library screeningScreening a genomic library by colony hybridization. Host bacteria transformed with a plasmid-based genomic library are plated on a petri plate and incubated overnight to allow bacterialcolonies to form. A replica of the colonies is obtained by overlaying the plate with a flexible disc composed of absorbent material (such as nitrocellulose or nylon).Find a piece of DNA that hybridizes with the probe. 1. Host bacteria with plasmid are plated and allowed to grow overnight2. A replica of the bacterial colonies is obtained on an absorbent dic3. The disk is treated with alkali (NaOH) and then brought back to neutral pH4. The disc is reacted with labeled probe5. Autoradiography of the disk reveals probe complementary DNA PCR1. dsDNA is denatured by raising temperature e2. specific pimers anneal to opposite strands of DNA. Primers provide 3’ OH for DNA polymerase 3. The temperature is raised to the working range of DNA polymerase The 3 steps to each cycle: denature DNA, anneal primers, extend both strands, repeat 20-30 timesThe reaction components:- DNA template ( or RNA in RT-PCR)- Two primers- Thermostable DNA polymerase- Mg 2+- dNTPs, buffer