Biomolecules and DNA Analysis

Forensics and DNA fingerprinting

Detecting Carbohydrates

• To detect monosaccharides and most disaccharides (reducing sugars) in stomach contents, a sample would be mixed with Benedict’s solution.

• A positive result is seen by the formation of a brownish-red cuprous oxide precipitate.

• You will test dextrose as a known for a positive result.

Detecting Carbohydrates

• Iodine is typically used to detect starch. It turns a dark blue/black in the presence of starch.

• You will test a known starch solution to see the result before testing the unknown “stomach contents.”

Detecting proteins

• Biuret solution is used to detect the presence of proteins.

• To observe the known reaction, you will mix biuret with albumin. A pinkish-purple color is observed if proteins are present.

Detecting Lipids

• Sudan III crystals are used to detect the presence of lipids.

• The procedure is important to note! A nonpolar solvent, like hexane is mixed with the sample and then poured off into an evaporating dish.

• The solvent must be completely evaporated before the crystals are streaked across the dish. A red streak is a positive test for lipids. No color change indicates no lipids are present.

Detecting Vitamin C

• The addition of 2,6-dichloroindophenol, which is a blue solution, turns light amber to colorless in the presence of ascorbic acid (vitamin C).

• You will test a solution of ascorbic acid to see the positive test.

• Then you should perform the test on the stomach contents to determine if vitamin C is present.

DNA

• Except for identical twins, no two people on earth have the same DNA.

• Since the 1980s, DNA has been used to solve crimes, identify victims, and establish paternity.

• It is especially useful in large-scale disasters, like plane crashes, hurricanes and tsunamis to identify victims.

Review of DNA

• DNA holds all the information needed to make proteins and to replicate itself.

• The genetic material is stored in chromosomes. If you dissolved the histone packaging, you would see the double helix strand of DNA tightly coiled around protein molecules and itself.

• If you could unwind the DNA, it would measure 6 feet in length!

DNA Structure

• DNA is composed of a sugar-phosphate backbone and ladder of nitrogenous base pairs.

• Adenine pairs with thymine

• Cytosine pairs with guanine

• There are 46 chromosomes in the nucleus of most cells (except sex cells…they have 23). This is called nuclear DNA.

• One chromosome pair is inherited from the mother and one from the father, so each person inherits exactly half of their genetic information from each parent.

• Mitochondrial DNA is in a circular loop and is inherited only from the mother. It is passed to the offspring in the cytoplasm of the egg cell.

Genes and Alleles

• Genes are DNA sequences that have instructions that determine our inherited traits.

• Examples: blood type, hair color, eye color, etc.

• An allele is one of two or more alternative forms of a gene. One allele comes from the mother and one from the father.

• Alleles are what is analyzed for dominant and recessive traits.

• The human genome is the total amount of DNA in the cell.

Coding and Noncoding genes

• The chromosomes in the nucleus of the cells contain 23, 688 encoded genes.

• This accounts for 1.5% of the DNA in the cell.

• The other 98.5% is noncoding and used to be referred to as junk DNA. However, more research is proving that even noncoding DNA is important in gene splicing.

Using DNA for Identification

• In the noncoding DNA, there is variation among humans. There are repeated base sequences that are unique to individuals.

• Certain sequences are repeated many times. These differences are referred to as polymorphism.

DNA Fingerprinting

• Dr. Alec Jeffreys determined that these repeating sequences could be isolated and analyzed. Thus, the beginning of DNA fingerprinting. (1984)

• Two types of repeating sequences are used: STRs (short tandem repeats) and VNTR (variable numbers of tandem repeats).

• These repeating sequences show up as different bands in electrophoresis separation techniques.

VNTR

• Certain short sequences of noncoding bases repeat multiple times.

• However, the number of times varies in each individual. One individual may have the sequence repeat 3 times while another is 7 times.

• Since the number of repeats varies, it is called variable number of tandem repeats (VNTR).

• The length of repeating bases in a sequence can vary from 9-80 bases.

STR

• The DNA sequences with a high degree of polymorphism are most useful for DNA analysis.

• These sequences are short- only 2-5 bases in length in the noncoding DNA.

• This method is useful by analyzing how many times the base sequence repeats in an individual.

• This is becoming the preferred method of analysis because the long VNTR sequences are harder to separate clearly.

DNA Profiles

• When several STR or VNTR sequences are examined, the DNA profile or fingerprint of a person is developed that is unique to that individual.

• This information is used in 2 ways:

• 1) Matching a tissue to see if it is from the same person. All bands match. (victim/perp ID)

• 2) Inheritance markings- since half of genetic material is from mother and half from father…half of the bands will align with a parent.

Sources of DNA

• Saliva, blood, seminal fluid, skin and hair may all contain DNA left at a crime scene.

• Since very small (or trace) amounts of DNA might be all that is available at the scene, a technique was needed to multiply the DNA to have enough to analyze.

• The use of the PCR (polymerase chain reaction) is employed to replicate the DNA samples

Collecting DNA

• 1) Wear disposable gloves and change them often.

• 2) Use disposable instruments for handling the sample (pipets, containers, etc.)

• 3) Avoiding touching, talking, coughing on, sneezing on areas where DNA is suspected to be.

• 4) Air-dry evidence before packaging in a paper envelope or bag.

• 5) If you cannot dry wet evidence before packaging, it can be frozen.

DNA Separation

• When the specific enzymes are used to cut the DNA in different fragments, the fragments are different sizes so they are different masses.

• The process of gel electrophoresis will allow them to be separated based on their mass.

• As more technology is developed, the different fragments are able to be separated by mass spectrometers based on their time of flight between charged plates.

DNA Fingerprinting

• 1) DNA must be extracted from its cells in the evidence sample.

• 2) DNA is “cut” by using restriction enzymes that recognize a unique base pattern in the DNA.

• 3) Amplification using the polymerase chain reaction (PCR).

• 4) Electrophoresis or other methods of separating fragments by mass. Smallest fragments move fastest through the gel.

Electrophoresis

• A gel, of aragose, is made which is porous.

• A buffer solution is placed in the gel tray. This aids the movement of the DNA based on differential charge.

• A power supply is attached and must be in contact with the solution. The DNA is placed in wells in the gel that is towards the negatively charged end of the gel.

• The DNA moves towards the positive charge. The smaller fragments move faster, and the larger fragments move more slowly. These create a band pattern that is unique for each individual.