Biogenetic Engineering

PCR Polymerase Chain Reaction (PCR) ·PCR is the cloning of DNA (amplification) ·Amount of DNA can be increased rapidly ·Useful if the source of DNA is small ·Temperature is used instead of enzymes (helicase) to separate the DNA strands ·Taq polymerase is thermostable (not denatured by the heat) and is still able to add new nucleotides to the parent strands ·This is an automated process that requires specialized machines called ‘Thermocyclers”

3 Steps to PCR Denature: Heat the solution containing the Taq polymerase, DNA and nucleotides to separate the double-stranded DNA into single strands Anneal: Cool the solution so primers can attach to the exposed complementary nucleotides on the single stranded DNA Extend: Once primers have attached to DNA, Taq polymerase adds nucleotides to both exposed primed DNA strands. Process is repeated many times…millions of copies in just hours

Animation: http://www.dnai.org/b/index.html Click techniques, then amplifying, then PCR animation

Gel Electrophoresis ·Agar gel with rectangular indentations called “wells” at one end between a positive and negative electrode. ·DNA that has been cut into fragments (by an enzyme) is mixed into a solution with dye and then placed into one of the wells ·An electrical current is passed across the gel.

Gel Electrophoresis ·DNA fragments are separated based on their size (how many base pairs long they are) and charge ·Since DNA is a negatively charged molecule all of the fragments will move towards the positive electrode (which is why the wells are always located nearest the negative electrode) - Smaller fragments are the easier to move through the gel and therefore will move more quickly to the right. - Large fragments move more slowly.

·The picture below is of a gel after the DNA has been separated ·Shows 6 separate samples of DNA ·Each band corresponds to a group of DNA molecules of the same size ·Samples b and c are identical and must therefore be from the same person

Uses Gel electrophoresis is used for DNA profiling ·Satellite (Tandem repeating) DNA are highly repetitive sequences of DNA from the non coding region of DNA. ·Different individuals have a unique length to their satellite regions. ·These can be used to differentiate between one individual and another. ·There are different types of 'DNA fingerprinting' for different circumstance - Forensic crime investigations - Parentage Issues - Animal breeding pedigrees - Disease detection

·Bands from different samples of DNA are compared to see if they match up ·In the case of forensic investigations, one sample would be from the crime scene, one from the victim, and one from the suspect ·If the bands match perfectly the suspect can be linked to the crime scene ·In the case to the right suspect #1 appears to have been at the crime scene (must still be proven by law to have committed the crime)

·In the example to the right Male #1 is the father ·In the case of paternity suits a sample of the child’s DNA, the mother’s DNA, and any potential father’s DNA is compared ·About half of the bands in the child’s DNA sample will match with the mother ·If the other half match with that of the third sample, the male is the father ·In the example to the right Male #1 is the father paternity animation http://www.sumanasinc.com/webcontent/animations/content/paternitytesting.html