DNA profiling or "fingerprinting" to analyse evidence in the law enforcement cases and other applications. Restriction Fragment Length Polymorphism has been theworkhorseof foreignsic DNA profiling for many years. Alec Jefferies in 1985, RFLP analysis provides a unique banding pattern of an individual's DNA sequence. DNA fragments are negativly charges and are drawn toward the positive pole (anode). Over a period of time, smaller DNA fragments travel farther than larger ones. These fragments of DNA in the gel will be stained and seen to provbide evidence to DNA matching. A quick analogy the desks and chairs have been pushed together. A individual student weaves through the desks no problem compared to where the strand of 4 students have a bigger difficulty taking more time.
The sililarities and the differences in DNA are used to show the specific nucleotide sequence and are shown in the gel when stained (see in pics). A radioactive complementary DNA probe can be recognized and binded; This probe can be described as "radio active lag" thaty will bind to a single stranded DNA fragment and produce a band in the gel. This brings together the lesson of "DNA fingerprinting" where the strands in the radiolabeled bands in the gel are used by determining the size of the DNA fragments in each band. Each person has a special bandings that will reflect the variations in the indivivuals' DNA.
These DNA evidence that is needed for DNA fingerprinting cna be obtained from any biological material and even dried material such as blood stains or mummified tissue.
Restriction Digestion of DNA
Restriction enzymes are the "chermical scissors" of the molecular biologist and the enzyme recognized a particular recognition sequence of the segment of DNA.
Making DNA Visible
Dna is colorless so the DNA fragments in the gel cannot be seen during electrophoresis which is a loading buffer containing two bluish dyes that is added to the DNA samples. The lodaing dyes doesnot stain the DNA itself but makes it easier to load the samples and monitor the progress of the DNA electrophoresis. The dye will "run towards red" just like the DNA fragments. The "faster" dye with the DNA will travel through the agarose gel and the slower ones will travel as well forming the now invisible streaks in the gel.
Reliability of DNA Evidence
In general one can assume that any two humans are 99.9% identical DNA sequence thus differing by only 1 bp in 1,000.
Alternative DNA Fingerprinting Scenarios
DNA finger printing and profiling also DNA typing are all names for the same process that shows relatedness of humans, pants or animals. Some of these that could be used for is Food idenification, Accused and convicted felons set free because of DNA typing, Identifyling human remains, Determining relatedness of humans, Studying relatedness amone ancient peoples, DNA testing families, Idenifying organisms that cause disease, Identifying birth parents, Proving paternity,Determining effectiveness of bone marrow transplants, Proving relatedness of immigrants, Confirming relatedness among animals, and DNA testing of plant material puts murderer at the scene.
Procedure:
put the tube containing the restriction enzyme on ice
label the tubes according to the instructions:
green CS
blue S1
orange S2
violet S3
red S4
yellow S5
label the tubes with name, date, period, table etc and put in tube holder
using the fresh tip for each sample pipet 10ml of each DNA sample from the stock tubes to transfer to the colored tubes
put 10micro liters into the bottom of the tubes to make sure not to suck it up
put the caps tightly on the the tubes and put onto a centrifuge and pulsate to collect all the liquid at the bottom
place the tubes in a water bath incubator for 45mins or overnight
when incubation period is done put in the refrigerator until the next lab period
DAY 2
remove the digested DNA samples from the refrigerator
use the centrifuge to bring all the liquid to the bottom of the tube
using a different tip for each sample put some loading dye into each tube and gently mix by contracting and retracting the pippet
remove the agarose gel from the refrigerator
place the agarose gel in the electrophoresis apparatus. fill with 1x TAE buffer to cover the gel
remember "run to red"
using a seperate tip for each tube put the samples into the agarose gel
put the lid on the elctrophoresis apparatus and run the power on 200 V for 12 mins
Visualization of DNA Fragments
when the run is complete turn off the power and remove the top of the chamber and remove the gel to put inot the box
Overnight Staining
add enough Fast Blast DNA stain to fill the top of the agarose gel and let stain overnight and put on the rocking table to make sure even staining.
drain the gel to make sure the gel is dry to be able to look at the bands the next day
Results and Observations:
Our group came to the consensis that Katie Records killed ME!!!! Also when the pippeting was done one of the time we may have contaminated one of the tubes but for some reason it didnt effect the results of the DNA finger printing.
Discussion:
a) I really had to idea who would have done it but I thought Mr.Chugh would pull a fast one and make it a crazy suicude and have me frame somebody. Also i had no idea how well this actually work in the forensic aspect of crime solving in the fields.
b) There could have been contamination in one of the tubes but it seemed to not affect any of the results thankfully.
