BCHM 4116 1st Edition Lecture 9 Outline of Last Lecture I Gibson Assembly II Gateway Cloning Recombination based cloning III IV V DNA library Construction Genomic DNA library construction Phage vector human DNA Making a cDNA library Outline of Current Lecture VI Methods VII Southern blot VIII Factors that determine the stringency of the Southern hybridization IX Introduction to Mutations X Northern blot XI Library screening XII PCR Current Lecture Methods Hybridization 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 probe Blotting 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 hybridization Similar 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 Blot 1 DNA is digested with restriction endonucleases 2 Gel electrophoresis is used to separate the fragments by size a The bands are visible under UV if soaked in ethidium bromide 3 The gel is then soaked in NaOH to denature and then add tris to neutralize 4 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 gel 6 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 visual Factors that determine the stringency of the Southern hybridization The 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 nonspecific 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 conditions Washing temp tm you get signal Washing 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 binding Low 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 tm High 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 C on 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 hybridization Information for probe design 1 Degenerate oligonucleotides 2 Heterologous probes 3 Fragment of gene of interest Labeling of the probe 1 Random priming and PCR are two of the many methods Northern blot This is the same except its done on RNA not DNA Gene 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 screening Screening a genomic library by colony hybridization Host bacteria transformed with a plasmidbased genomic library are plated on a petri plate and incubated overnight to allow bacterial colonies 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 overnight 2 A replica of the bacterial colonies is obtained on an absorbent dic 3 The disk is treated with alkali NaOH and then brought back to neutral pH 4 The disc is reacted with labeled probe 5 Autoradiography of the disk reveals probe complementary DNA PCR 1 dsDNA is denatured by raising temperature e 2 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 times The reaction components Visual Two primers Thermostable DNA polymerase Mg 2 dNTPs buffer DNA template or RNA in RT PCR