What is it? The Human Genome Project was an effort to determine the complete sequence of DNA in the human genome. Its goal was to discover and map all of the approximately 35,000 human genes and make them available for further biological study.
Techniques used: Restriction Fragment Length Polymorphisms Automated DNA Sequencing Polymerase Chain Reaction
Restriction Fragment LengthPolymorphisms (RFLPs) Each restriction enzyme is specific to a certain base sequence, a “restriction site”, and will cut up DNA at all such sites to produce a number of “restriction fragments”. No one will have the exact same base sequence unless they are identical twins. Because of the DNA variability, restriction fragments from a given region of an individual’s genome can be separated using get electrophoresis to reveal a unique pattern (a finger print). Inheritance of RFLPs can be followed through families. By using the RFLPs scientists can create linkage maps.
Step 1: Isolate the DNA To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane. Appropriately separate the DNA from other cell components. When doing so, make sure that the process doesn’t damage the DNA at all.
Step 2: Restriction Digestionand Gel Electrophoresis The extracted DNA is digested with specific restriction enzymes. Each restriction enzyme will recognize and cut up DNA in a predictable way, resulting in a reproducible set of DNA fragments, or restriction fragments, or different lengths.
Step 2: Restriction Digestion and GelElectrophoresis (continued) The millions of restriction fragments produced are commonly separated by electrophoresis on agarose gels.
Step 3: Transfer DNA bySouthern Blotting The gel is denatured in a basic solution and placed in a tray. A porous nylon or nitrocellulose membrane is laid over the gel, and the whole thing is weighted down. All the DNA restriction fragments in the gel are transferred as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.
Step 4: DNA Hybridization Themembrane with the target DNA is incubated with the DNA probe.
DNA Probe The DNA probe usually comes from a DNA library, which is a collection of vectors that contain a representation of an original DNA molecule cut into pieces. Vectors may be transformed into bacteria and may multiply the piece of DNA they contain many times. The DNA probe is also converted into a single- stranded molecule, conveniently labeled, using any standard method.
Step 4: DNA Hybridization(continued) If strands on the membrane are complementary to those of the probe, hybridization will occur and labeled duplexed formed. If strands are highly stringent, hybridization with distantly related or non-homologous DNA does not happen. The DNA probe picks up sequences that are complementary and ideally homologous to it among the thousands or millions of undetected fragments that migrate through the gel.