Biotechnology Part 2

How do forensic scientists determine who’s blood has been left at a crime scene? How are paternity tests conducted? ANSWER: Using restriction enzymes, a technique called Gel Electrophoresis, and a concept called RFLP (restriction fragment length polymorphism)

Individuals have differences in their DNA sequences. Differences in coding regions (exons) may result in a mutation (ie. Sickle cell anemia)

RFLP analysis If a sample of DNA is digested by a restriction enzyme, the result would be the formation of a number of DNA fragments of various lengths. Since we all have different DNA, exposure to the same restriction enzyme would produce different numbers and lengths of fragments for each individual This is known as RESTRICTION FRAGMENTS LENGTH POLYMORPHISM (RFLP)

RFLP can be used to identify individuals. Scenario: A crime is committed and there are 3 suspects. If the criminal left a sample of blood at the crime scene, RFLPs from this sample can be compared to RFLPs from blood of all suspects to determine who the criminal is.

Gel Electrophoresis After restriction enzymes have been used to cut DNA, DNA fragments must be separated and purified from each other for analysis using a process called gel electrophoresis In this process, the DNA fragments are placed in wells on a gel called agarose (agar) An electrical current is passed through the gel and since DNA is negatively charged, the fragments of DNA are attracted to the positive end.

As the DNA fragments move through the agar, the small fragments move quickly, but larger fragments move slowly because they get caught up in the gel Therefore, smaller fragments will travel further away from the negative electrode A blue dye is added initially to the DNA fragments. When the dyed fragments reach the positive end, the power is turned off.

Once the gel electrophoresis is complete, a fluorescent dye is then used to stain the DNA fragments. Ideally this creates a band pattern that is unique to each individual.

In reality, the amount of DNA is usually so large and the bands too numerous that instead of seeing individual bands, a large smear is seen on the gel. In a process called Southern blotting, the DNA from the gel can be transferred from the agarose to a nylon membrane.

SOUTHERN BLOTTING - sUMMARY The gel is subjected to a chemical that causes the double-stranded DNA to be denatured into single-stranded DNA Single stranded DNA is transferred to a nylon membrane (this is the blotting). The nylon membrane is places on the gel with a positive electrode behind it to attract the negative DNA The nylon is placed in a solution with radioactive probes (complimentary nucleotides) that will bind to specific regions of the DNA that have been chosen by the scientist (areas of mutation etc.)

The nylon membrane is placed against an X-ray film The probes are radioactive and cause exposure of the X-ray film This pattern imprinted on the X-ray film is called an autoradiogram When the film is developed, a pattern will emerge…

Match the Suspect The band pattern of suspect 1 matches the specimen. Thus, suspect 1 is probably our criminal The specimen must also be compared to the victim because the victim’s blood may be mixed up in the specimen or it could just be the victim’s blood.

You are NOT the father! IN paternity test, the child’s DNA is compared to its mother’s and the possible fathers. Since the child has DNA from both its mom and dad, the bands that match its mother can be ignored. Look at the bands that do not match the mother’s Who does it match? The reason the child doesn’t have the exact same DNA of its parents is because it only receives half of each parent’s chromosomes/DNA

PCR: Polymerase Chain Reaction PCR is a technique used to clone (amplify) DNA Before the technique was developed, it took a very long time to make a copy of a DNA sequence A scientist would have to place the gene into a plasmid and then wait for the bacterial cell to make more copies during replication The scientist would then have to cut the plasmid and remove the DNA sequence of interest

With PCR, many copies of DNA can be made quickly This is particularly useful when only a small sample of original DNA is available (Ex: If only a small sample of DNA is obtained from a crime scene, PCR may be used to allow for multiple forensic tests.)

Before running a DNA through the Gel Electrophoresis for analysis, the sample will undergo PCR to make sure there is enough DNA for adequate testing The process of PCR is closely related to DNA replication that occurs within the nucleus of a cell.

DNA REPLICATION The 2 DNA strands are separated using enzymes helicase and gyrase PCR The 2 strands of DNA are separated using heat At temps of 94°C – 96 °C, the hydrogen bonds between the complimentary strand will break, separating the strands

DNA REPLICATION An RNA primer must be added first before DNA nucleotides are added to build a complementary strand to the template strand PCR A DNA primer is used instead because they are easy to produce in labs. One of the primers is known as a forward primer and the other is a reverse primer cause they start synthesis of DNA in opposite directions The temp must be lowered to 50°C - 65°C in order for the primers to anneal to the template DNA

PCR Taq polymerase – a type of DNA polymerase – builds complementary strands using DNA nucleotides that have been added to the solution The Taq polymerase does not become denatured because it is extracted from bacteria that live in hot springs DNA REPLICATION Once the RNA primers have been laid down, DNA polymerase III will build a complementary strand by adding DNA nucleotides

When the complementary strands have been built, the cycle can repeat itself over and over again, doubling the number of copies each time.

Simplified process….