DNA Fingerprinting Review

Why DNA? DNA is individual evidence DNA links or eliminates a suspect to a crime DNA identifies a victim even if no body is found or if the body is decomposed

DNA Basics Genetic information is stored in molecules of DNA making up structures called chromosomes Humans have 23 pairs of chromosomes

DNA DeoxyriboNucleic Acid – Determines an organism’s traits. – Found in the nucleus of a cell. – Looks like a twisted ladder.

DNA Structure DNA is made of repeating subunits called nucleotides. Nucleotides have three parts: – Simple sugar – Phosphate group – Nitrogen base “ Backbone ”

DNA STRUCTURE Phosphate Group Sugar (Deoxyribose) Nitrogen Base Nucleotide DNA has a negative charge because of the phosphate ions in its chemical “backbone.”

DNA Structure Four possible Nitrogen Bases: – Thymine (T) – Adenine (A) – Cytosine (C) – Guanine (G) Nitrogen Bases pair up: – Adenine and Thymine (A-T) – Cytosine and Guanine (C-G)

Where Is DNA Found? DNA is found in all nucleated body cells: – white blood cells – semen and saliva – bone and tissue – hair root and teeth Most abundant in buccal (cheek) cells. Red blood cells have NO nuclei; and therefore, no DNA.

DNA Typing “Fingerprinting” Only one-tenth of 1% of DNA (about 3 million bases) differs from one person to the next. We have unique patterns of repeating base sequences Scientists use these regions to generate a DNA profile of an individual.

DNA Replication PCR: Polymerase Chain Reaction – A technique used for making copies of a defined segment of a DNA molecule. – Can be valuable when you don’t have a lot of evidence from the crime scene. – Millions of copies of DNA can be made from a single speck of blood or a few skin cells. – Allows for highly degraded samples of DNA to be analyzed.

How does PCR work? 1.Heat the DNA strands- causes the strands to separate (unzip). 2.Cool the mixture and add a primer- a short sequence of base pairs that will add to its complementary sequence on the DNA strand. 3.Add a DNA polymerase (large protein) to recombine the separated strands. 4.Heat again to around 75° C for the strands to recombine.

Step 1: Separate DNA strands Step 2: Add base pairs to make complimentary sequences (A-T and C-G) Step 3 and 4: DNA Polymerase grabs strands and recombines them

PCR – What’s the point? The outcome is a doubling of the number of DNA strands. Heating, cooling, and strand rebuilding is repeated 25 to 30 times = more than one million copies of the original DNA molecule. Each cycle takes less than two minutes from start to finish.

DNA Typing 1. RFLP: Restriction Fragment Length Polymorphisms a)Isolate: separate DNA from the cell Use lysis solution b)Cut: to make shorter base strands A restriction enzyme will recognize a specific sequence of bases and cut the DNA at a specific point that can then be separated and characterized for identification.

DNA Typing 1. RFLP: Restriction Fragment Length Polymorphisms c)Sort: by size (small to large) Use electrophoresis d)Analyze: look at specific alleles for identification

DNA Typing Electrophoresis: A technique used to separate DNA fragments. – An electrical current is moved through a gel substance causing molecules to sort by size. *Remember that DNA has a negative charge!! – The smaller, lighter molecules will move the furthest on the gel. – After developing, the fragments can be visualized for characterization.

Electrophoresis

2. Prepare DNA samples in centrifuge 1. Pipet DNA into tubes

Electrophoresis 4. Run the gel using electric current. 5. Observe and compare bands of DNA. 3. Load DNA into the gel wells.

DNA Typing 2. STR: Short Tandem Repeats – STR’s are locations (loci) on the chromosome that contain short sequences of 2 to 5 bases that repeat themselves in the DNA molecule. – The DNA is not cut!!! – The advantages of this method are that it provides greater discrimination, requires less time, a smaller sample size, and the DNA is less susceptible to degradation.

Outcomes to DNA Fingerprinting Match: DNA profiles appear to be the same. (Lab will determine the frequency). Exclusion: The genotype comparison shows profiles that could only have originated from two different sources. Inconclusive: The data does not support a conclusion as to whether the profiles match.

CODIS Combined DNA Index System – DNA profiles uploaded to this national database – Run by the FBI – Used for linking serial crimes and unsolved cases with repeat offenders – Links all 50 states

Is there DNA in urine? Urine itself does NOT contain DNA, but it may contain epithelial cells (which do contain DNA). – Most healthy individuals do not excrete epithelial cells in their urine.

RFLP’s – DNA sample is broken into pieces (digested) by restriction enzymes. – The resulting restriction fragments are separated according to their lengths by gel electrophoresis.

Types of DNA Nuclear  Found in the nucleus  Consists of 23 pairs of chromosomes inherited from both parents  Each cell contains only one nucleus Mitochondrial  Found in the cytoplasm  Is inherited ONLY from your mother  Can be found in skeletal remains  Used when nuclear DNA is not available

Uses of DNA Profiling  To identify potential suspects  To exonerate individuals  To identify crime victims  To establish paternity  To match organ donors