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

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D. Creating a DNA Profile RFLP Method - Restriction Fragment Length Polymorphism RFLP (often pronounced "rif lip", as if it were a word) is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. RFLPs can be used in many different settings to accomplish different objectives. Some uses include in paternity cases or criminal cases to determine the source of a DNA sample, to determine the disease status of an individual, or to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans. RFLP Production Each organism inherits its DNA from its parents. Since DNA is replicated with each generation, any given sequence can be passed on to the next generation. An RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between. A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs. A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. When a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is since the probe is detectable. RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence. For example, let's follow a particular RFLP that is defined by the restriction enzyme EcoR I and the target sequence of 20 bases GCATGCATGCATGCATGCAT. EcoR I binds to its recognition seuqence GAATTC and cuts the double-stranded DNA as shown: In the segement of DNA shown below, you can see the elements of an RFLP; a target sequence flanked by a pair of restriction sites. When this segment of DNA is cut by EcoR I, three restriction fragments are produced, but only one contains the target sequence which can be bound by the complementary probe sequence(purple). 44
Let's look at two people and the segments of DNA they carry that contain this RFLP (for clarity, we will only see one of the two stands of DNA). Since Jack and Jill are both diploid organisms, they have two copies of this RFLP. When we examine one copy from Jack and one copy from Jill, we see that they are identical: Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)--- GAATTC- Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)--- GAATTC- When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics). Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT-- (1.3 kb)-GAATTC- Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT-- (1.3 kb)-GAATTC- Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the other's in molecular weight: 45
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 and 42: 2. cDNA is denatured at more than 9
Page 43: SYBR-green SYBR green is another me
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
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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

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

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