Restriction Fragment Length Polymorphism (RFLP)

Size: px
Start display at page:

Download "Restriction Fragment Length Polymorphism (RFLP)"

Transcription

1 Restriction Fragment Length Polymorphism (RFLP)

2 Polymorphism is any difference in the DNA sequence between individuals. Since we are all genetically different from each other, we are all polymorphic. This difference is exploited in RFLP analysis. RFLP requires a large sample of DNA which is fresh. The whole genome is required, a segment of DNA is not enough. RFLP is often called genetic fingerprinting and is often used How does it work? by law enforcement to link evidence left at a crime scene with the DNA of a suspect.

3 Step 1. A large sample of DNA is extracted from a tissue sample. The DNA is cut up by one or more restriction enzymes. This results in DNA fragments of various sizes which are run on a gel (gel electrophoresis) to produce smears. (invisible at this point). restriction enzyme gel electrophoresis Whole DNA DNA fragments smears

4 Step 2. Southern blotting is performed. A chemical is added to the gel to separate the double stranded DNA. The resulting single-stranded DNA is then transferred (blotted) onto a nylon membrane using an an electrical eletrical charge. Safety_Managment/Pouring.gif + - Chemical separates strands nylon membrane with single stranded DNA. single stranded DNA on gel

5 Step 3. The nylon membrane is then immersed in a solution containing radioactive complimentary nucleotide probes which bind to specific regions of DNA fragments. This is called hybridization. nylon membrane with single stranded DNA (invisible). solution containing radioactive probes.

6 Step 4. The nylon membrane is then placed against x-ray film. Over a period of 2-3 weeks the radioactive probes expose the film in areas of hybridization. hybridiztion. The pattern of dark bands that is produced is called an autoradiogram and is unique to each individual. nylon membrane with radioactive probes (invisible) x-ray film

7 Step 4. The nylon membrane is then placed against x-ray film. Over a period of 2-3 weeks the radioactive probes expose the film in areas of hybridization. hybridiztion. The pattern of dark bands that is produced is called an autoradiogram and is unique to each individual. Figure 3 pg. 299

8 PCR and RFLP PCR RFLP Table 1 pg. 300 State of sample tiny and degraded large and fresh Size of sample target sequence the whole genome Time required 1 day about 3 weeks Basic premise building complementary strands using the principles of DNA replication cutting DNA using restriction enzymes; using radioactive probes Result medium gel autoradiogram Tools Sensitivity and accuracy Taq polymerase, nucleotides, DNA primers, gel electrophoresis sensitive and accurate restriction enzymes, radioactive probes, nylon membrane, X-ray film, gel electrophoresis highly sensitive and accurate

9 PCR and RFLP When these techniques are used in criminal investigation, not only can they result in the conviction of the guilty, but also the exoneration of the innocent. In 1984, Guy Paul Morin was arrested and served 10 years for the murder of a 9 year old girl. Guy Paul Morin Based on DNA evidence (in this case PCR), Mr. Morin was acquitted of the crime in The actual killer has yet to be found.

10 Read pgs Do #6-8,10-14 pg. 300