Genetic Research and Biotechnology Gel Electophoresis, Polymerase Chain Reaction, and, Dideoxysequencing And STR (short tandem repeats)

Gel Electrophoresis Gel electrophoresis (real life) Uses of gel electrophoresis: DNA fingerprinting (animation) Separating pieces of DNA to isolate them Estimating the size of DNA molecules

Dideoxysequencing (aka the Sanger method) Basic overview This method is used to determine the sequence of bases in a strand of DNA. What is a ddntp? https://www.youtube.com/watch?v=e2G5zx-OJIw How does the process work with using gel electrophoresis? https://www.youtube.com/watch?v=vK-HlMaitnE https://www.youtube.com/watch?v=FvHRio1yyhQ

Dideoxynucleotides

The Method DNA denatured into single strands using heat. Radioactive primer is attached to the template strand. The solution is divided into four tubes labeled "G", "A", "T" and "C". Reagents added as follows: "G" tube: all four dNTP's, ddGTP and DNA polymerase "A" tube: all four dNTP's, ddATP and DNA polymerase "T" tube: all four dNTP's, ddTTP and DNA polymerase "C" tube: all four dNTP's, ddCTP and DNA polymerase 5. Polymerase is then allowed to copy the DNA sequence

Building the New DNA As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymerase. Whenever a dideoxynucleotide is added to the chain the DNA sequence ends.

In the “G” tube we might find the following mixture:

Separating the Pieces of new DNA Once these reactions are completed, the DNA is denatured in preparation for electrophoresis. The contents of each of the four tubes are run in separate lanes to separate the different sized bands from one another. Smaller fragments migrate faster across the gel. If all of the reactions from the four tubes are combined on one gel, the actual DNA sequence in the 5' to 3' direction can be determined by reading the banding pattern from the bottom of the gel up. It is important to remember that this sequence is complementary to the template strand you are trying to sequence.

Automated Sequencing More DNA can be sequenced in a shorter period of time. The reactions are performed in a single tube containing all four ddNTP's, each labeled with a different color dye. As in Sanger's method, the DNA is separated on a gel, but they are all run on the same lane as opposed to four different ones.

STR (short tandem repeats) Most of our DNA is identical to the DNA of others However there are regions of our DNA that can vary from person to person The human genome is full of repeating sequences of 2 – 6 bp long (like a genetic stutter) They are found mostly near the centrome of chromosomes STR’s are very useful for DNA analysis in forensic cases and paternity testing Mr Andersen on STR’s https://www.youtube.com/watch?v=DbR9xMXuK7c