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Transcript of RFLP-DNA Fingerprinting
Allows to visually see DNA bands
uses an electric current to separate different-sized molecules in a porous, sponge-like matrix
smaller molecules move more easily through the gel pores than larger molecules
DNA flows from negative cathode to positive cathode
phosphate has a negative charge
RFLP stands for "restriction fragment length polymorphism"
Acts as a molecular marker
Locus specific and may be co-dominant, with both alleles in a heterozygous sample being detected
RFLP sample hybridizes with the DNA sample after separation by electrophoresis
In order to make accurate comparisons between the crime scene DNA and each suspect DNA, each DNA band was measured (in millimeters) from the beginning of the well to the center of each band. These comparisons are then used to decipher which suspect committed the crime.
Stained with Ethidium Bromide
binds tightly to the DNA double helix, and glows when illuminated with ultraviolet light
The purpose of the "DNA fingerprinting" experiment was to detect and figure out which Suspect would be considered criminal by comparing DNA fragments of Suspects 1, 2, 3, 4 and 5 to DNA fragment of the Crime Scene.
As the result, the bands of RFLP DNA fragments of the Suspect 3 matched most close to the DNA bands of the Crime Scene. Therefore, Suspect 3 would be considered criminal.
recognizes the palindromic sequence setup “GAATTC” to break the base pairs and denature the hydrogen bonds
leaves 'sticky ends' on the pieces of viral DNA it chops up.
called sticky because they are available to hybridize with complementary sequences.
makes staggered rather than blunt cuts, and leaves short chains of single nucleotides hanging off the ends of severed molecules.
Recognizes the site “CTGCAG” to break the sugar-phosphate bond and begin hydrolysis
Produces ends with 3’ overhangs, rather than blunt cuts
Adding the restriction endonuclease
The tubes of the DNA samples from the crime scene and suspects 1-5 were collected from the front which contained 10 micro liters each. 10 micro liters of the enzyme mix, "ENZ", was added to each one. The ENZ enzyme mix is added to "cut" the DNA molecule.
Based on Jim Harvey's speech structures
Analyzing The Gel
After the gel had ran, the gel had to be analyzed. First, the gel was put under UV light and then a picture was taken. With this picture, each band was measured from the well to the center of each DNA band in mm. The HindIII digest well created a standard in which the DNA can be measured by fragment size. By matching up exact matching fragments, the crime scene can be matched with a suspect, as shown in the Data.
The measurements for distance traveled were graphed along the x-axis of a graph with the y-axis representing base pair numbers.
A particular measurement for distance traveled corresponds with an approximation of base pair number in reference to the digest marker.
Hind III Digest
"deoxy" means that there is a hydrogen molecule instead of an –OH group on the secondary carbon of the five-carbon sugar
consists of a series of nitrogenous base molecules held together across the molecule by weak hydrogen bonds, and linearly by strong phosphodiester bonds.
After comparing the distance traveled to the base pair numbers, it was found that Suspect 3 had the closest DNA match to that of the Crime Scene.
Differences in the measurements of the bands from the Crime Scene versus Suspect 3 are 0.10mm for each of the three bands of DNA measured. Base pair differences between the Crime Scene DNA and Suspect 3 are 800bp, 100bp, and 70bp respectively.
So, if Suspect3 is the "closest match", what does that mean?
Adenine pairs with Thymine
-2 hydrogen bonds
Cytosine pairs with Guanine
-3 hydrogen bonds
Since Suspect 3 is the closest match, that means the linear sequence of DNA is most similar to that found at the crime scene.
RFLP detects differences and similarities between the 4 nitrogenous bases - A, T, C, G - from the restriction endonuclease cuttings sites.
Discovered by Dr. Werner Arber and Dr. Hamilton Smith in 1968
Specifically break the sugar-phosphate bonds when added to the DNA recognition sites, causing hydrolysis
Known as molecular scissors or “cutting” enzymes
EcoR1 and PstI = Type II restriction enzymes
cut DNA at specific nucleotide sequences
What about the measurements for the other suspects?
CS vs. S4
Difference in Distance Traveled: 0.20mm, 1.20mm, 0.50 mm
Base Pair number difference: 700bp. 2000bp, 100bp
RFLP is an important method in genome mapping, determination of gene locations for genetic disorder, and paternity testing.
inexpensive, easy to design, and no requirement for expensive instruments
some restriction enzymes are expensive
difficult to identify exact variation that SNPs affect restriction enzyme recognition site.
requirement of large amounts of hand-on-time.
Real World Use
The first DNA profiling technique inexpensive enough to see widespread use
Also used for genome mapping, determination for risk of disease, paternity testing, or breeding patterns in animals
Bio-Rad, "Introduction to DNA Fingerprinting". 23-46
Griffiths, Anthony J.F., et al. "Introduction to Genetic Analysis". 10th edition. 2010. 138.
National Center for Biotechnology Information. Probe – Reagents for Functional Genomics. Restriction Fragment Length Polymorphism. Web. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml>
Thermo Scientific. PstI. Web. <http://www.thermoscientificbio.com/restriction-enzymes/psti>
Purines and Pyrimidines < http://www.bio.miami.edu/tom/courses/bil255/bil255goods/09_dna.html>
DNA Base Pairing < http://www.phschool.com/science/biology_place/biocoach/bioprop/basepair.html>
Structure of DNA < http://www.genome.gov/multimedia/illustrations/FactSheet_DNA.pdf>
Gel Electrophoresis < http://www.web-books.com/MoBio/Free/Ch9C.htm>
Ethidium Bromide < http://www.mun.ca/biology/scarr/IG1_16_14.jpg>
1 x TAE electrophoresis buffer
-usually at pH 8.0
-sequesters divalent cations
After a week of incubation, the DNA samples were ready to be ran on a gel electrophoresis. To keep track of the electrophoresis, 5 micro liters of loading dye were added to each sample (C1-S5) and then mixed. The gel was loaded into a gel box and then had electrophoresis buffer poured until it covered the top.
The following RFLP-DNA Fingerprinting procedure will use an enzyme mix of both EcoR1 and PtsI.
The hypothesis "
If the DNA fragments of the suspects are correctly cut by the restriction endonuclease, then the correct suspect can be identified by way of measuring the DNA bands and comparing the measurements with that of the crime scene
failed to be rejected.
If the DNA fragments of the suspects are correctly cut by the restriction endonuclease, then the correct suspect can be identified by way of measuring the DNA bands and comparing the measurements with that of the crime scene.
The HindIII lambda digest, which is a DNA standard, was acquired by the professor. This already had loading dye in it and none additional had to be added. The samples were loaded starting with the HindIII Lambda digest and then went up from C1 to S5 respectively in the wells. 20 microliters were loaded in every well except for the HindIII well which only had 10 microliters. The gel was ran for 30 minutes at 125 volts.
Gel Electrophoresis Continued
Restriction fragment length polymorphism (RFLP) technology
is applied to analyze different base-pair sequences of DNA sample of Suspects by using restriction endonuclease enzyme called EcoR1.