MBIO 3410 lecture Oct 2 2007

MBIO 3410 lecture Oct 2 2007 - Last Lecture Types of...

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1 Last Lecture Types of restriction modification systems Type I Type II Type II restriction enzyme activity Type III Recognition sequences Cohesive ends Restriction enzymes Other modification systems Dam methylation Dcm methylation Mcr system DNA ligation Restriction digests & gel analysis Separating DNA fragments Agarose gel electrophoresis Making an agarose gel DNA migration Molecular mass (length) Shape Staining DNA gels Restriction digests
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2 Pulsed-field Gel Electrophoresis
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3 Pulsed-field Gel Electrophoresis Pulsed-field gel electrophoresis (PFGE) is used to resolve extremely large DNA molecules This method uses altered direction of the electric current to alter the path of DNA molecules as they travel through the gel The upper size limit of DNA separation in agarose gels is raised to well over 10 Mbp (10 000 kbp) If the DNA is forced to change direction during electrophoresis, different sized fragments within the diffuse unresolved DNA band begin to separate from each other The advent of PFGE meant that single DNA molecules representing whole chromosomes could be separated with ease using gels There are two main types of PFGE: Field inversion gel electrophoresis (FIGE) contour-clamped homogeneous electric field (CHEF) electrophoresis
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4 PFGE a) Current is applied across the gel for a defined period called the pulse time In the range of 0.5–2 min The direction of the current is then switched b) The repetitive switching of the current means that the DNA will zig- zag down the gel The zig-zagging motion allowed the separation of large DNA molecules c) Using homogenous electric fields, straight-line separation patterns are obtained, like the separation of whole yeast chromosomes
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5 Several parameters act together during PFGE to affect the effective separation range of a particular gel Type of agarose used Concentration of agarose used Buffer composition Buffer temperature Electric field strength Reorientation angle Pulse time Pulse time is primarily responsible for changes in the effective separation range Shorter pulse times lead to separation of shorter DNA molecules the smaller DNA fragments will begin to move more quickly upon reorientation than the larger fragments. Longer pulse times lead to separation of larger DNA molecules
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6 The effect of pulse time on the separation of DNA fragments of different sizes The chromosomes of the yeast S. cerevisiae and a set of known molecular size DNA markers were subjected to PFGE under otherwise identical conditions using different pulse times Increasing the pulse time gives rise to better separation of large DNA fragments Figure 2.19 The sizes of the markers are shown in kbp
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7 Field inversion gel electrophoresis (FIGE) To obtain straight lanes field inversion gel electrophoresis (FIGE) uses parallel electrodes to assure an homogeneous electric
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This note was uploaded on 07/05/2008 for the course MBIO 3410 taught by Professor Richardson during the Fall '07 term at Manitoba.

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MBIO 3410 lecture Oct 2 2007 - Last Lecture Types of...

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