Lecture 22- Biotechnology III: RFLPs and Southern Blot Flashcards
What is a polymorphism?
A difference in the DNA sequence of different individuals. They can be as small as one base pair.
How can restriction enzymes and polymorphisms be used to differentiate individuals?
Because of differences in the DNA (polymorphisms), adding the same restriction enzymes to the DNA of two different persons will lead to different-sized fragments of DNA.
What can RFLP (Restriction Fragment Length Polymorphisms) be used for?
For example, taking a sample of DNA from a dad and his son, the sizes of the fragments obtained when applying restriction enzymes should be the same if they are in fact related.
How can we know if someone has a specific gene?
We use the southern blot.
How does the southern blot work?
First, the DNA samples are isolated and cut up using restriction enzymes. Then, the fragments are run through a gel to separate them based on their size. Finally, a radioactive probe made of base pairs complementary to those we are looking for is added. If the sequence we are looking for is present, the probe will be bound to it and will be detected under x-ray.
What are ddNTPs and why are they useful?
They are di-deoxyribose triphosphates. These base pairs lack the 3’ OH, this makes them useful as they terminate an extension reaction (polymerase can’t add any more base pairs next to it). The attachment of a ddNTP puts a stop to a PCR reaction.
How does DNA sequencing work with ddNTPs?
A sample of DNA is amplified using PCR, and the fragments are divided into 4 tubes. Each tube contains all base pairs in excess and one ddNTP (either ddATP, ddGTP, ddTTP, or ddCTP).
In the tube with ddCTP, when comes times for the polymerase to add a C, it will either add a C with a 3’ OH, or a ddCTP, which stops the reaction.
At the end, we are left with fragments of each size (1,2,3,4,5, etc base pairs). We can work backwards because we know the last base pair of each fragment.
1 bp fragment tells us the first base pair, 2 bp fragment tells us the 2nd base pair, etc.
How are the fragments of different sizes separated when sequencing DNA?
Using electrophoresis and a gel.
