Chapter_7_-_Finding_the_Right_Clone_v2

Chapter_7_-_Finding_the_Right_Clone_v2 -...

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7.1 Screening Libraries with Gene Probes To find a gene of interest, it is common to use a DNA/RNA probe which will hybridize to the DNA sequence of interest that you are looking for in a library The probe is hybridized to DNA-binding membrane filters which contain a replicate of the colonies or plaques of the gene library
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7.1 Screening Libraries with Gene Probes 7.1.1 Hybridization Weaker hydrogen bonds separate between the bases separating the two strands when DNA is denatured , and is reversible ( renaturation ) DNA strands can be separated by heat or chemically (NaOH), and often referred as melting During melting, the optical density ( absorbance ) changes (at 260nm ) as the double stranded DNA separates The midpoint of DNA melting is the melting temperature (T m ) T m varies according to the DNA base composition Melting temperature increase with the GC content (3 bonds) versus the AT (2 bonds) Figure 7.1 Melting (denaturation) of DNA
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7.1 Screening Libraries with Gene Probes 7.1.1 Hybridization – Hydrophobic interactions T m can be approximated using the formula : T m = 2 o C x (number of [A+T] + 4 o C x (number of [G+C]) Hydrophobic interactions affect the DNA stability and T m : between adjacent bases ( stacking ) on the same strand between the bases on opposite strands Hydrophobic nature of the bases means that a single-stranded molecule is unstable, and wants to prevent exposure to water and tend to stick together non-specifically Specificity of interactions can thus be increased by chemicals (such as formamide) that reduce hydrophobic interactions In single stranded RNA/DNA, nuceic acis form secondary structures such as hairpins and stem-loops
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7.1 Screening Libraries with Gene Probes 7.1.1 Hybridization – Ionic strength Negative charge on the phosphate groups in the nucleic acid backbone causes electrostatic repulsion of the two strands Negative charge on DNA works in opposition to the hydrophobic interactions In presence of salt, cloud of counterions neutralizes the negative charge on the phosphate groups, weakening the electrostatic repulsion forces By reducing the salt concentration, weak interactions between the strands will be disrupted by electrostatic repulsion, and increase the specificity of hybridization
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7.1 Screening Libraries with Gene Probes 7.1.1 Hybridization – formation of hybrid DNA Mixed DNA fragments of partial similarity will hybridize together upon renaturation The lower the stringency , (like a lower melting temperature) allows greater numbers of mismatches of hybridized fragments Raising the salt concentration, allows more mismatches because there is fewer counterions relieving the repulsion between strands (ie. Lowers stringency) Salt concentrations are usually regulated as multiples of SSC solution (1x,2x,3x Standard Saline Citrate) Formamide raises the stringency by
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_7_-_Finding_the_Right_Clone_v2 -...

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