Biotech Continued….Biotech Continued….

4.44.4

How do forensic scientists determine How do forensic scientists determine who’s blood has been left at a crime who’s blood has been left at a crime scene?scene?

How are paternity tests conducted?How are paternity tests conducted?

ANSWER: Using restriction enzymes, a ANSWER: Using restriction enzymes, a technique called Gel Electrophoresis, technique called Gel Electrophoresis, and a concept called RFLP (and a concept called RFLP (restriction restriction fragment length polymorphismfragment length polymorphism))

Individuals have differences in their Individuals have differences in their DNA sequences.DNA sequences.

Differences in coding regions (exons) Differences in coding regions (exons) may result in a mutation (ie. Sickle may result in a mutation (ie. Sickle cell anemia)cell anemia)

Differences in noncoding regions Differences in noncoding regions (introns) may exist in the form of (introns) may exist in the form of variations in the number of repeating variations in the number of repeating unitsunits

RFLP analysisRFLP analysis

If a sample of DNA is digested by a If a sample of DNA is digested by a restriction enzyme, the result would be the restriction enzyme, the result would be the formation of a number of DNA fragments of formation of a number of DNA fragments of various lengths.various lengths.

Since we all have different DNA (because we Since we all have different DNA (because we all have different numbers of VNTR) all have different numbers of VNTR) exposure to the same restriction enzyme exposure to the same restriction enzyme would produce different numbers and would produce different numbers and lengths of fragments.lengths of fragments.

This is known as This is known as RESTRICTION FRAGMENTS RESTRICTION FRAGMENTS LENGTH POLYMORPHISMLENGTH POLYMORPHISM

RFLP can be used to identify RFLP can be used to identify individuals.individuals.

Scenario: A crime is committed and Scenario: A crime is committed and there are 3 suspects.there are 3 suspects.

If the criminal left a sample of blood at If the criminal left a sample of blood at the crime scene, RFLPs from this the crime scene, RFLPs from this sample can be compared to RFLPs sample can be compared to RFLPs from blood of all suspects to from blood of all suspects to determine who the criminal is.determine who the criminal is.

Gel ElectrophoresisGel Electrophoresis The DNA fragments must be separated and The DNA fragments must be separated and

purified from each other for analysis using a purified from each other for analysis using a process called process called gel electrophoresisgel electrophoresis

In this process, the DNA fragments are In this process, the DNA fragments are placed in wells on a “jello-like gel- called placed in wells on a “jello-like gel- called agarose (or agar)agarose (or agar)

The agarose is fibrous and acts like a netThe agarose is fibrous and acts like a net An electrical current is passed through the An electrical current is passed through the

gelgel Since DNA is negatively charged, the Since DNA is negatively charged, the

fragments of DNA are attracted to the fragments of DNA are attracted to the positive end.positive end.

As the DNA fragments move through As the DNA fragments move through the agar, the small fragments move the agar, the small fragments move quickly, but larger fragments move quickly, but larger fragments move slowly because they get caught up in slowly because they get caught up in the fibres of the gel.the fibres of the gel.

A blue dye is added initially to the A blue dye is added initially to the DNA fragments. When the dye DNA fragments. When the dye reaches the positive end, the power reaches the positive end, the power is turned off. is turned off.

A fluorescent dye is then used to A fluorescent dye is then used to stain the DNA fragments.stain the DNA fragments.

Ideally this creates a band pattern Ideally this creates a band pattern that is unique to each individual.that is unique to each individual.

Molecular Markers are DNA fragments of known lengths. They may be run during a gel electrophoresis so that the fragments of the sample DNA can be compared to the markers to determine approximate length.

In reality, the amount of DNA is usually so In reality, the amount of DNA is usually so large and the bands too numerous that large and the bands too numerous that instead of seeing individual bands, a large instead of seeing individual bands, a large smear is seen on the gel. smear is seen on the gel.

In a process called SOUTHERN BLOTTING, In a process called SOUTHERN BLOTTING, the DNA from the gel can be transferred to the DNA from the gel can be transferred to a nylon membrane.a nylon membrane.

The nylon membrane is placed against an The nylon membrane is placed against an X-ray film to read an autoradiogram which X-ray film to read an autoradiogram which will show the band patternwill show the band pattern

Match the SuspectMatch the Suspect The band pattern of The band pattern of

suspect 1 matches the suspect 1 matches the specimen. Thus, suspect specimen. Thus, suspect 1 is probably our criminal1 is probably our criminal

The specimen must also The specimen must also be compared to the be compared to the victim because the victim because the victim’s blood may be victim’s blood may be mixed up in the mixed up in the specimen or it could just specimen or it could just be the victim’s blood.be the victim’s blood.

Who’s your Daddy???Who’s your Daddy??? IN paternity test, the child’s DNA is IN paternity test, the child’s DNA is

compared to its mother’s and the compared to its mother’s and the possible fathers. Since the child has possible fathers. Since the child has DNA from both its mom and dad, the DNA from both its mom and dad, the bands that match its mother can be bands that match its mother can be ignored.ignored.

Look at the bands that do not match Look at the bands that do not match the mother’sthe mother’s

Who does it match?Who does it match?

The reason the child doesn’t have The reason the child doesn’t have the exact same DNA of its parents is the exact same DNA of its parents is because it only receives half of each because it only receives half of each parent’s chromosomes/DNAparent’s chromosomes/DNA

PCR: Polymerase Chain PCR: Polymerase Chain ReactionReaction

PCR is a technique used to clone PCR is a technique used to clone (amplify) DNA(amplify) DNA

Before the technique was developed, Before the technique was developed, if scientists wanted to make multiple if scientists wanted to make multiple copies of a gene fragment, the gene copies of a gene fragment, the gene would have to be inserted into a would have to be inserted into a plasmid and the bacterial cell would plasmid and the bacterial cell would make more copies when it replicates make more copies when it replicates its plasmids. Then scientists would its plasmids. Then scientists would have to remove the plasmids and cut have to remove the plasmids and cut out the bacterial genes.out the bacterial genes.

With PCR, many copies of DNA can be With PCR, many copies of DNA can be made quickly.made quickly.

This is particularly useful when only a This is particularly useful when only a small sample of original DNA is available.small sample of original DNA is available.

(Ex: If only a small sample of DNA is (Ex: If only a small sample of DNA is obtained from a crime scene, PCR may be obtained from a crime scene, PCR may be used to allow for multiple forensic tests.)used to allow for multiple forensic tests.)

Before running a DNA through the Gel Before running a DNA through the Gel Electrophoresis for analysis, the Electrophoresis for analysis, the sample will undergo PCR to make sure sample will undergo PCR to make sure there is enough DNA for adequate there is enough DNA for adequate testing.testing.

The process of PCR is closely related The process of PCR is closely related to DNA replication that occurs within to DNA replication that occurs within the nucleus of a cell.the nucleus of a cell.

DNA REPLICATIONDNA REPLICATION

The 2 DNA strands The 2 DNA strands are separated are separated using enzymes using enzymes helicase and helicase and gyrasegyrase

PCRPCR

The 2 strands of The 2 strands of DNA are separated DNA are separated using heatusing heat

At temps of 94At temps of 94°C°C – – 96 96 °C°C, the , the hydrogen bonds hydrogen bonds between the between the complimentary complimentary strand will break, strand will break, separating the separating the strandsstrands

DNA REPLICATIONDNA REPLICATION

An RNA primer An RNA primer must be added must be added first before DNA first before DNA nucleotides are nucleotides are added to build a added to build a complementary complementary strand to the strand to the template strandtemplate strand

PCRPCR

A DNA primer is used A DNA primer is used instead because they are instead because they are easy to produce in labs.easy to produce in labs.

(The temp is lowered to (The temp is lowered to 5O°C - 65 °C to allow for to allow for the DNA primers to the DNA primers to attach) attach)

One of the primers is One of the primers is known as a forward primer known as a forward primer and the other is a reverse and the other is a reverse primer cause they start primer cause they start synthesis of DNA in synthesis of DNA in opposite directions opposite directions

DNA REPLICATIONDNA REPLICATION

Once the RNA Once the RNA primers have been primers have been laid down, DNA laid down, DNA polymerase III will polymerase III will build a build a complementary complementary strand by adding strand by adding DNA nucleotidesDNA nucleotides

PCRPCR

Temp raised to Temp raised to 76°C

Taq polymerase – a Taq polymerase – a type of DNA type of DNA polymerase – polymerase – builds builds complementary complementary strands using DNA strands using DNA nucleotides that nucleotides that have been added have been added to the solutionto the solution@ 76°C – the Taq polymerase does not denature because it is extracted from a bacteria that lives in hot springs (Used to hot temps!)

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

Simplified process….Simplified process….

AnimationsAnimations

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