What are the steps of DNA fingerprinting?

5 Comments

1. ?Melody? says:

   September 9, 2009 at 6:01 am

   DNA fingerprinting is a laboratory procedure that requires six steps:

   1: Isolation of DNA.
   DNA must be recovered from the cells or tissues of the body. Only a small amount of tissue – like blood, hair, or skin – is needed. For example, the amount of DNA found at the root of one hair is usually sufficient.

   2: Cutting, sizing, and sorting.
   Special enzymes called restriction enzymes are used to cut the DNA at specific places. For example, an enzyme called EcoR1, found in bacteria, will cut DNA only when the sequence GAATTC occurs. The DNA pieces are sorted according to size by a sieving technique called electrophoresis. The DNA pieces are passed through a gel made from seaweed agarose (a jelly-like product made from seaweed). This technique is the biotechnology equivalent of screening sand through progressively finer mesh screens to determine particle sizes.

   3: Transfer of DNA to nylon.
   The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight.

   4-5: Probing.
   Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint. Each probe typically sticks in only one or two specific places on the nylon sheet.

   6: DNA fingerprint.
   The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously. It resembles the bar codes used by grocery store scanners.
2. N E says:

   September 9, 2009 at 6:01 am

   Extract DNA from a sample
   Subject the DNA to restriction enzymes
   Separate the different size fragments by subjecting the restriction enzyme-treated DNA to agarose gel electrophoresis
   Transfer the DNA to a nitrocellulose or nylon membrane
   Expose the membrane to a radioactively labelled DNA probe
   Expose X-ray film to the membrane and develop the film

   This is also called restriction fragment length polymorphism (RFLP) analysis.

   Here are a couple of links:

   http://www.reachoutmichigan.org/funexperiments/agesubject/lessons/newton/dna.html
   http://www.pbs.org/wgbh/nova/sheppard/analyze.html

   http://www.accessexcellence.org/RC/AB/BA/DNA_Fingerprinting_Basics.html
3. dlm says:

   September 9, 2009 at 6:01 am

   Materials:
   40 ul. EcoRI
   20 ul. BamHI
   20 ul. HindIII
   120 ul. lambda DNA (0.5 mg/ml)
   200 ul. 2x compromise restriction buffer
   20 ul. loading dye
   0.8% agarose (4 g. agarose/ 500 ml. TBE buffer)
   5 liters 1X TBE Buffer
   Carolina Blue stain
   50 microcentrifuge tubes
   10 0.5 – 10 ul. micropipettors and tips
   37oC water bath
   10 sets electrophoresis equipment
   10 microtube racks
   hot plate with magnetic stirrer or microwave oven
   KEEP IN MIND THAT THE DNA SAMPLES IN THIS LAB ARE ACTUALLY DIFFERENT RESTRICTION ENZYMES AND THE RESTRICTION ENZYME IS ACTUALLY LAMBDA DNA!!

   DNA sample tubes for suspects X, Y, and Z as well as from blood samples collected at the scene of the crime (E) should be prepared as follows:

   suspect X DNA: 20 ul. HindIII (X)
   suspect Y DNA: 20 ul. EcoRI (Y)
   suspect Z DNA: 20 ul. BamHI (Z)
   evidence DNA: 20 ul. EcoRI (E)
   (2 ul. aliquots in 1.5 ml tubes may be supplied to students to save time and materials)

   The 120 ul. of l DNA (0.5mg. / ul. ) should be aliquoted 12 ul. per lab group and be labeled "restriction enzyme" (R).

   The 200 ul. of compromise restriction buffer should be aliquoted 20 ul. per lab group and labeled "buffer" (B).
   We used the BamHI buffer as our compromise buffer and had good results.

   0.8% agarose may be prepared by combining 4 grams of agarose with 500 ml. TBE buffer, microwave, and stir until suspended. Heat and keep at 60oC using water bath. Allowing 40-50 ml. per gel, this should be enough to pour at least 10 gels.
   Analysis:

   DNA profiling, or as it is more commonly called, DNA fingerprinting, involves three basic steps– restriction of the DNA samples into fragments that can be handled more easily: separation by size of the various length fragments using electrophoresis; and then visualizing certain fragments to which radioactive probes have been attached.

   A review of DNA fingerprinting in general would be appropriate at this time. Keep in mind that this lab only simulates an actual DNA fingerprint, and so the final step using probes will not be done.

   Compare the DNA profiles of the suspects in this case to the DNA profile of the blood found at the crime scene. Which suspect does this test put at the crime scene?
4. Kinase says:

   September 9, 2009 at 6:01 am

   1. Cell lysis (open up the cells) and extraction of DNA.
   2. Purify the DNA. Remove all excess cell debris.
   3. Quantify the DNA. (figure out how much DNA is present) This step is VERY important. You need to know this so that you use the correct amount of sample for step 4.
   4. You can use restriction enzymes on this next step, but usually not necessary. Usually the next thing you will want to do is PCR (polymerase chain reaction)…essentially copy the sequence(s) of DNA interested in.
   5. Run DNA copies on a gel (electrophoresis) or a 310 analyzer using probes.
   6. Visual analysis of the results…here you will do scientific interpretation of the gel or 310 printout.
5. Kaylan says:

   April 12, 2011 at 8:32 pm

   I am doing a Independent Study project in my Quest class. On Apps that help the FBI and CIA and so if any body has info on NA Fingerprinting or Apps please post back and help.