RFLP Analysis Restriction Fragment Length Polymorphism 1.Extract/Isolate DNA from the cell restriction enzymes 2.Cut DNA into smaller fragments using restriction enzymes electrophoresis 3.Sort fragments by size using electrophoresis radioactive probes 4.Visualize the specific fragments for comparison using radioactive probes

STEP 1. Extract DNA from cell Break cell membrane – release nucleus Open nucleus - release DNA protect DNA from enzymes Separate from other cell parts DNA must be precipitated un-dissolved from solution

Step 2. Cut DNA into fragments Restriction Enzymes Found in bacteria cells Many different kinds Read from 5’  3’ (can NOT read DNA both ways)

Each restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides called a restriction site.

5’..TAAGCTACGGAGTTAGCTCCGTAA..3’ 3’..ATTCGATGCCTCAATCGAGGCATT..5’ How many cuts would this particular enzyme (Alu I) make in this segment of DNA?

5’..TAAGCTACGGAGTTAGCTCCGTAA..3’ 3’..ATTCGATGCCTCAATCGAGGCATT..5’ How many cuts would this particular enzyme (Alu I) make in this segment of DNA? 2

restriction enzyme ‘X’ + 46 chromosomes w/ lots of enzyme ‘X’ restriction sites = many cuts many fragments of DNA

Step 3. Sort DNA fragments by size A thick gel matrix is used Agarose Gel or Polyacrylamide Gel Like a sponge with nooks and crannies Electrophoresis

Load DNA into the gel wells. Pipette DNA

 Run the gel.  An electrical current is moved through a gel substance causing molecules to sort by size.  The smaller, lighter molecules will move the furthest on the gel.

Step 4. Visualization of Fragments Sooooo many fragments in the human genome Can NOT just add a dye or stain to all DNA Would result in a continuous smear. Southern Blot Analysis label and photograph only the specific fragments of DNA that you are comparing

1)DNA transferred to nylon membrane radioactive DNA probe 2)Add radioactive DNA probe - that binds to complimentary DNA sequence Autoradiography 4) Autoradiography - X-ray film applied to membrane  expose/visualize bands that are attached to probes DNA Fingerprint Result is the DNA Fingerprint Southern Blot Analysis

If we follow the same procedure for each person’s DNA, how does this process result in different DNA banding patterns? Why does each person have a unique set of fragment sizes?

C G A G G A A T C T T C T T C T T C T C A G G A T G G A T A G G A T C Restriction Site (A G / G A) C G A G G A A T C T T C T T C T T C T C A G G A T G G A T A G C A T C C G A G G A A T C T T C T T C T C A G G A T G G A T A G G A T C Insertions / Deletions – minor differences/mutations at restriction site STR’s & VNTR’s Differences in numbers of tandem repeats Everyone has a different amount of each specific restriction site scattered throughout their DNA. Even though our DNA may be cut at the same sites, there may be more tandem repeats between two sites. C