In situ Detection Methods; DNA homology
Geneticists have invented methods to detect the unique DNA sequence in a colony of bacteria “in situ” or directly in the bacteria cell. The detection of DNA sequence takes advantage of a property of double stranded DNA that was discovered by E.M. Southern, the inventor of the Southern Blot. The main idea is that DNA molecules that have similar sequences can anneal to each other and form stable, detectable DNA hybrids. If you intend to clone a gene and a similar gene has already been cloned, this screening method is a logical strategy.
The best way to understand “in situ” hybridization of DNA is to walk your way through the procedure. The starting point in library screening will be a plate of bacterial colonies from your gene library. A replicate is made of this plate full of colonies in case the procedures get messy. From the plate, the following steps are performed.
1) Place a membrane that will bind DNA or proteins over the plate of colonies. Some cells from each colony will adhere to the membrane. Care is taken to align the membrane so that the top of the membrane is at the top of the plate. Then the original colony can be located on the plate.
2) Dip the membrane in a solution that will break open the bacteria cells. The protein and DNA in the cells will then bind to the membrane. The double stranded DNA is also denatured so that single strands of DNA will be bound to the membrane surface.
3) The membrane is placed into a solution with milk proteins to block all of the surfaces that are not bound with the proteins and DNA from the bacteria. This allows the bacterial protein and DNA to be the only target for the next step.
4) The membrane is placed into a bottle or bag with a hybridization solution.
Now the DNA sequence can be detected on the membrane by using the clone of a similar gene. The next procedures turn this similar gene into a hybridization probe.
5) Copies of the similar cloned gene are made by performing replication of the DNA in a test tube. The replicated copies will have special nucleotides incorporated that can be detected such as radioactive nucleotides or nucleotides with a digoxigenin attached.
6) The DNA copies are heated to make single stranded DNA.
7) The single stranded DNA is added to the bag or bottle with the membranes (step 4 above). Now the copies of the similar gene act like a “probe” and seek out single stranded DNA molecules bound to the membrane that have complementary sequences. The probe will stick if the sequences of the probe and membrane-bound DNA complement well enough.
8) Unstuck probe is washed off and the bound probe is detected with either an x-ray film (when radioactive nucleotides or chemiluminescence are used) or antibody detection system (when digoxigenin nucleotides are used).
If the screening method works, a signal will be detected where the probe has found and bound the DNA from one or a few colonies in the library. Then a few living bacteria from the corresponding colony on the replicate plate can be selected, grown and they will make clones of the gene of interest.