Investigating CSI – Background material Table of Contents I ...

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This process is repeated many times. After 30 to 40 rounds of synthesis of cDNA, the reaction products are usually analyzed by agarose gel electrophoresis. The gel is stained with ethidium bromide to visualize the cDNA. An agarose gel stained with ethidium bromide and illuminated with UV light which causes the intercalated stain to fluoresce. This type of agarose gel-based analysis of cDNA products of reverse transcriptase-PCR does not allow accurate quantitation since ethidium bromide is rather insensitive and when a band is detectable, the logarithmic stage of amplification is over. This problem will be addressed in more detail below. Ethidium bromide is a dye that binds to double stranded DNA by interpolation (intercalation) between the base pairs. Here it fluoresces when irradiated in the UV part of the spectrum. However, the fluorescence is not very bright. Other dyes such as SYBR green, which are much more fluorescent than ethidium bromide, are used in real time PCR. 42
SYBR-green SYBR green is another method to monitor DNA synthesis. SYBR green is a dye that binds to double stranded DNA, but not to single-stranded DNA, and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does). We also use SYBR green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of singlestranded DNA is much higher that the ratio for ethidium bromide. Other methods can be used to detect the product during real-time PCR. Sybr-green fluoresces brightly only when bound to double-stranded DNA There are many real time machines available and the one shown below is the ICycler ® from BioRad. The lid slides back to accommodate samples in a 96-well plate format. This means that we can look at a lot of samples simultaneously. The machine contains a sensitive camera that monitors the fluorescence in each well of the 96-well plate at frequent intervals during the PCR Reaction. As DNA is synthesized, more SYBR Green will bind and the fluorescence will increase. source: http://www.med.sc.edu:85/pcr/realtime-home.htm 43
Page 1 and 2: Investigating CSI - Background mate
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Page 5 and 6: A forensic scientists needs a honou
Page 7 and 8: lond, is also wearing Air Jordans w
Page 9 and 10: of unidentified remains with the pr
Page 11 and 12: Matching DNA Proving that a suspect
Page 13 and 14: Creating a DNA Profile: The Basics
Page 15 and 16: with thymine and guanine with cytos
Page 17 and 18: power is the combined analysis of a
Page 19 and 20: of DNA profile comparisons done by
Page 21 and 22: II. Tools and Techniques for DNA Fo
Page 23 and 24: p30 Test Unlike the presumptive aci
Page 25 and 26: Specificity refers to how many othe
Page 27 and 28: expected to occur when using your c
Page 29 and 30: helps to bind oxygen as well.) When
Page 31 and 32: chemical reaction, but in order to
Page 33 and 34: 2. Brauner, P.& Gallili, N., "A Con
Page 35 and 36: DNA Extraction Techniques Organic-b
Page 37 and 38: C. Quantification of DNA Southern B
Page 39 and 40: Southern Up Close In this simplifie
Page 41: 2. cDNA is denatured at more than 9
Page 45 and 46: Let's look at two people and the se
Page 47 and 48: at this locus and a different sized
Page 49 and 50: These loci are located on the same
Page 51 and 52: Short Tandem Repeat (STR) Analysis
Page 53 and 54: The other two PowerPlex16 groups of
Page 55 and 56: The Federal Bureau of Investigation
Page 57 and 58: eference sample from the standard s
Page 59 and 60: III. Interesting Cases Using DNA Ev
Page 61 and 62: me. But I was also lucky that when
Page 63 and 64: The tests indicated that the drops
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Page 69 and 70: But only 18 of the states are hooke
Page 71: that to all felony offenses, as Ala

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