ࡱ> 3527 bjbjUU "27|7|l )$; [R @mFEF0)Summary emmy Munoz >From: "Emmy Munoz" >To: miss_emmy_13@hotmail.com >Subject: summary >Date: Tue, 24 Oct 2006 02:51:13 +0000 > > >Capillary electrophoresis (CE) is a relatively new technique of >electrophoresis, with its first use in the 1980s.CE offers many >advantages over conventional slab gel electrophoresis. The entire CE >process can be automated, from injection and separation to detection. >Also, only small amounts of sample are used up in the CE process, leaving >enough samples for retesting. Other advantages of CE include: faster >sample processing and the ease of detection and interpretation of results. >The disadvantages of CE include its small throughput capacity. Only one >sample can be run at a time, so processing many samples will take longer >than slab gel electrophoresis. However, some newer instruments permit >running several samples at a time. The costs of CE instruments are >considerably higher than slab gel instruments. >The components of the CE instrument include: a narrow capillary, two >buffer vials, two electrodes connected to a power source, a laser >excitation source, fluorescence detectors, a auto sampler to hold sample >vials and a computer component that controls sample injection and >detection. The capillary is made of fused silica and contains gel used to >separate the DNA molecules. Detection in the CE instrument is automatic >and the computer generates readout. >Separation of DNA in electrophoresis relies on the different sizes of DNA >passing through the gel. Smaller DNA molecules are eluted first because >they are able to travel through the gel pores faster. Larger molecules are >retarded more than smaller molecules. Two models of DNA separations have >been described: the Ogston sieving model and reputation. The Ogston model >regards DNA as a spherical shaped molecule running through pores in the >gel. Larger molecules are not able to go through the pores and will not >get through the gel. Reputation regards DNA as a large, snake-like >molecule running through the gel with separations due to the winding of DNA >through the gel. > > > > >Inman ch6 Pg 83-95 > >Length Polymorphisms > >Pcr can be used to amplify short polymorphisms. The procedures for all >three detection systems are similar. The PCR product is loaded into a gel >but this gel is made out of a different substance called polyacrylamide. >This gel is more appropriate for analyzing the small size PCR products. The >fragments are separate by length. The bands are stained directly. A silver >stain is used to visualize the separated DNA bands and the gel is then >dried to be kept as a permanent record. Just like RFLP each locus will >produce one or two bands representing the alleles present. STRs are now >most commonly analyzed using florescent detection and automated analysis. >These results are called electropherograms, which are presented as a series >of peaks. One major advantage of fluorescent detection is that only one of >the two denatured strands from each DNA duplex is visualized. Because the >resolution power of the gel system is so high. In fluorescent detection, >only the primer complementary to one strand is tagged, thus eliminating any >confusion resulting from reading doublets at each allele. Additionally, the >use of multiple colored fluorescent tags allows the combination of STR loci >in which the lengths of some overlap. These can be run in the same gel lane >and still be clearly distinguished by color. > >_Ch12 Need for DNA separation Typical separation size range from 100-400bp Separation method be reproducible and have results that can be compared by other labs DNA separation by electrophoresis in stab-gel or capillary environment Electrophoresis Phosphate group on DNA has negative charge DNA molecules migrate away from negative electrode Movement of ions generate heat, heat must be dissipatedexcessive heat can cause gel to fall apartcapillaries can dissipate heat better Stab Gels Stab gels consist of series of wells and a buffer solution that DNA moves through during electrophoresis Agarose gels RFPL uses agarose gels to separate fragments in sizes of ~600bp to ~23000bp Low molecular weight DNA not separated well w/ agarose gels PCR amplifies STRs (~100bp to ~400bp) better separated by polyacrylamide gels Agarose is a seaweed, contains pores 200nm in diameter Each wells generally can hold 5-10 microliters of sample Samples are mixed with a loading dye (bromophenol blue) to help see the sample 8-24 samples are run at a time on a agarose gel Anode (+ electrode) farthest away from wells 100-600 volts Smaller DNA molecules move faster than larger ones When separation is completed, gel is scanned or photographed Just to let you know Emmy, Cary and Tony turned in the ch 12 summary ________________________________________________________________ de()WX ! ~  ; < ? @ $QV 9>qv mr<Aqvxyz{|}~CJOJQJaJo(CJOJQJaJW 1h/ =!"#$% i8@8 NormalCJ_HaJmH sH tH <A@< Default Paragraph Font20  DJ  y ,0Xfryeljl +33333 CC:\Documents and Settings\Steven Lee\Desktop\Summary emmy Munoz.doc@hB@@UnknownGz Times New Roman5Symbol3& z Arial[ monospaceTimes New Roman"qhɪfɪfJ"202Summary emmy Munoz  Oh+'0l   ( 4 @LT\dSummary emmy Munozumm mmmmmmNormal  rm1rmMicrosoft Word 9.0@F#@ E@.CEJ՜.+,0 hp|   r" Summary emmy Munoz Title  !#$%&'()+,-./014Root Entry F@bpFE61TableWordDocument"2SummaryInformation("DocumentSummaryInformation8*CompObjjObjectPool@bpFE@bpFE  FMicrosoft Word Document MSWordDocWord.Document.89q