BIOTECHNOLOGY PART 2

How are paternity tests conducted? ANSWER: How do forensic scientists determine whose 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)

Polymorphism Individuals have differences in their DNA sequences. any difference in DNA sequence (coding or non-coding) that can be detected between individuals Polymorphisms can occur in both coding regions and non-coding regions

Polymorphism Example 1 – polymorphisms in coding regions: Organisms that are the same species carry the same genes, but may have different alleles for those genes (blue eyes vs. brown eyes) Can also be used to detect mutations (someone with sickle cell anemia will have a different allele for one of the hemoglobin proteins which gives the rbc a sickle shape)

Polymorphism Example 2 – polymorphisms in non-coding regions: Non-coding regions of DNA often contain sequences in which base pairs repeat over and over again (these are known as variable number tandem repeats or microsatellites) Variations in these microsatellites exist between individuals of the same species

RFLP ANALYSIS Therefore, different individuals will have variations (polymorphisms) between their DNA sequences SO?? Assume … we have two samples of DNA (from different individuals) we use a restriction enzyme to cut these DNA samples The DNA is cut up into pieces which are called restriction fragments

RFLP ANALYSIS Since there are differences in the sequences, the restriction enzymes will not cut in the exact same places Therefore, different individuals will produce restriction fragments of different lengths This is known as restriction fragment length polymorphism (RFLP)

Example 1: SAMPLE 1 SAMPLE 2 When separated, Sample 1 will produce a very different banding pattern than sample 2. Hence the 2 can be differentiated.

Example 2: Both DNA samples are cut with the same restriction enzyme The enzyme does not cut in the exact same places since there is variation in their DNA sequences

These fragments can then be run on a gel (using a technique called gel electrophoresis) which separates the fragments according to length

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RFLP analysis 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.

Gel Electrophoresis 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 (fibrous matrix) 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.

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

Let’s re-examine our previous example: Which is the positive end and which is the negative end?

Consider a different example: Chromosome A contains a sequence detected by a restriction enzyme

Gel Electrophoresis 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…

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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 (polymerase chain reaction) 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

PCR: Polymerase Chain Reaction 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.)

PCR: Polymerase Chain Reaction Before running a DNA sample 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.

PCR: Polymerase Chain Reaction The PCR procedure requires primers, DNA polymerase and DNA nucleotides. The DNA polymerase used is from a bacteria called Thermus aquaticus. This bacteria is a thermophile and thrives at high temperatures (lives in hot springs) The DNA polymerase of this species is known as Taq polymerase and it functions optimally at about 70 degrees Celsius Taq polymerase is used because it does not denature at the high temperatures needed for the PCR procedure

PCR: Polymerase Chain Reaction http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.html

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 because 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

DNA REPLICATION Once the RNA primers have been laid down, DNA polymerase III will build a complementary strand by adding DNA nucleotides PCR Taq polymerase – a type of DNA polymerase – builds complementary strands using DNA nucleotides that have been added to the solution

PCR: Polymerase Chain Reaction

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….

PCR: Polymerase Chain Reaction http://www.youtube.com/watch?v=eEcy9k_KsDI