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

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Running the Gel An electric current is passed through the gel and the DNA moves away from the negative electrode. The distance moved depends on the size of the DNA fragment. Standards are used to quantitate the size. Unrestricted, large DNA runs as a smear due to random shearing (breaking) of the DNA. The DNA can be visualized by staining first with a fluorescent dye and then lighting with UV. Transfer of the DNA to a Membrane The DNA is first denatured (made single stranded - usually by raising the pH) and then transferred out of the gel and onto a membrane. The transfer can be done electrically or by capillary action with a high salt solution. Development of the blot A radioactively-labeled probe specific for the gene in question is incubated with the blot. The blot is washed to remove non-specifically bound probe and then a development step allows visualization of the DNA that is bound. See below for a detailed description of this process. In this example the gene is found in the second largest fragment of the restricted DNA. In the unrestricted DNA it migrates more slowly because it is part of a larger molecule. To use this method to quantify DNA it is necessary to compare your results to a standard curve. You would need to use a smaple that contains a known quantity of DNA. 38
Southern Up Close In this simplified cartoon, two different DNAs (single stranded following denaturation) are bound to a membrane. The membrane is incubated with a solution containing a probe (another single stranded DNA or RNA, or oligonucleotide) which is homologous to one of the two DNAs on the membrane. The probe has an indicator attached to it so that it may be detected later on. The binding is very specific and requires the base pairs of the probe and the bound DNA to be perfectly complementary, i.e. A to T and C to G or for RNA; A to U and C to G. A wash step removes any probe which is not tightly bound to the DNA on the membrane. Only DNA that matches exactly will remain bound. In this example, DNA "A" is the specific DNA homologous to the probe and therefor a band will develop where this DNA is bound to the membrane. No band will show where "B" is bound source: http://www.escience.ws/b572/L22/south.html 39
Page 1 and 2: Investigating CSI - Background mate
Page 3 and 4: convict the killer of Gill Dando wa
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: C. Quantification of DNA Southern B
Page 41 and 42: 2. cDNA is denatured at more than 9
Page 43 and 44: SYBR-green SYBR green is another me
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
Page 65 and 66: July 17, 1918. Czar Nicholas II, hi
Page 67 and 68: Guest list pared With the refusal o
Page 69 and 70: But only 18 of the states are hooke
Page 71: that to all felony offenses, as Ala

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

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