Forensic SciencePresents

DNA

A. Terminology

1. Chromosomes –are strands of genetic material

2. Genes – fundamental unit of heredity; they instruct cells to make proteins

• Locus (loci) exact location on the DNA molecule of a gene or area or inerest.

• 4. Homozygous – two identical gene pairs

5. Recombinant DNA—opening up base pairs of the helix and recombining it with another strand

7. Genetic code– sequence of letters on a DNA strand

8. Restriction enzymes—chemicals that cut DNA into fragments that can later be incorporated into another DNA strand; about 150 are commercially available.

9. probe—a single strand of nucleic acid, much like RNA, that has been made in a way that its base sequence lines up to hybridize areas on an allele; usually labeled with radioactive material

10. Human genome—a project (13 year) designed to determine the order of bases on all 23 pairs of human chromosomes.

• The project is now complete. • Knowing where on a specific

chromosome DNA codes for a particular protein is useful for diagnosing and treating genetic diseases.

B. History

1. James Watson and Francis Crick—in 1953 discovered the configuration of the DNA molecule.

2. Alec Jeffreys– the first to recognize DNA is unique to everyone. He isolated DNA markers and called them DNA “fingerprints.”

3. Kary Mullis – in 1985 developed PCR, or polymerase chain reaction testing

C. Structure

1. polymer– a very large molecule made by linking together a series of repeating units.

• DNA is a polymer, so is a protein, cellulose, polyethylene. The repeating units are called monomers.

http://http://www.rsc.org/ej/DT/2000/b003769i/b003769i-f44.gif

2. Nucleotides– the “monomers” of DNA; consist of a 5 carbon sugar, a phosphate group, and a nitrogenous base.

3. The bases of DNA are:

4. DNA is a double-helix; that means it has two coiled strands.

5. Base pairing– a purine must pair with a pyrimidine, therefore:

Adenine pairs to thymine andGuanine pairs to cytosine

6. Proteins – made by linking together a combination of amino acids. There are 20 known amino acids.

. Amino acid codes-building blocks of protein are coded by a sequence of 3 bases.

D. Replication

1. Process—unwinding the DNA strand in the double helix; exposing the strand to a collection of free nucleotides; letter by letter the double helix is recreated in the proper order.

2. Polymerases—enzymes that assemble a new DNA strand in the proper base sequence determined by the original or parent DNA strand.

E. DNA Typing

1. RFLP—restriction fragment length polymorphism; requires high molecular weight DNA. What are they for?

There are repeating fragments of DNA that do not code for proteins and can be used for human ID.

The RFLP enzymes cut these out.

a. Visual evaluation--Soak and free up the DNA

b. Assay– electrophoresisc. Digestion-by the restriction enzymed. Test gele. Prepare known samplesf. Electrophoresis of all samples-smaller

fragments move at a faster rate

g. Southern blotting-transfer the fragments to a nylon membrane. (Named after its developer, Edward Southern.)

h. hybridization-nylon treated with radioactive probes containing a base sequence complementary to the RFLP’s being identified.

i. autoradiography-a nylon sheet is placed against x-ray film and exposed for several days.

j. Additional probes-adding more probes increases the probability of having a match.

2. PCR-Polymerase Chain Reaction

Needs only low-molecular weight DNA

a. DNA is denatured by heating it to 95°C, splitting the bonds between the two halves.

b. Heat is reduced and DNA primer are added which hybridizes to site-specific arrangements of complementary bases.

d. Step C is repeated over and over

e. DQA1—gene used in PCR testing that contains a number of variations among humans

3. Short Tandem Repeats (STR)

a. advantage—higher discrimination that RFLP and reduces the time to get results

b. Location- on the chromosome that contains short sequence elements that repeat themselves within the DNA molecule

c. Length-3 to 7 bases

d. TH01– a STR on a gene that repeats the sequence A-A-T-Ge. multiplexing—using a variety of STR’s in order to further

narrow down the sample1. importance—increases the probability of being

only from one source2. frequency of occurrence—gets smaller with the

number of STRs you can run.f. Process of capillary electrophoresis– two ends of a capillary

tube are placed in a buffer. The column is coated with a gel polymer and the DNA is injected into one end. The other end of the column is connected to a detector that tracks the separated STRs. STR fragments with then move through the column when an electrical current is added.

g. Amelogenin gene1. chromosome location—sex chromosomes X and Y2. body location—tooth pulp3. male—will show two bands4. female—will show only one band5. difference importance—this particular gene will at least show a sex difference even when a perfect match is impossible.

F. Mitochondrial DNA

1. location—in the cytoplasm (in the mitochondria, baby.)

2. inheritance- solely from the mother3. Function of mitochondria—gives

energy to the cell in the form of ATP

4. Importance – more mitochondria can be found in one cell so you don’t need as much

5.Difference between mtDNA and nuclear DNA testing – MtDNA is more rigorous, time consuming and more costly$$$

6. Testing process – mtDNA is found in circles or loops instead of linear strands. HV1 and HV2 have the most variation in humans. These areas are generated through PCR, and the order of bases is then determined.

7. Case of the Unknown Soldier – mtDNA was used with 7 different families to determine that remains belonged to First Lt. Michael Blassie who was shot down in Vietnam in 1977.

8. Significance – Blassie’s family is from Missouri.

G. Collection and System ID of DNA

1. CODIS – Combined DNA Index System; a national system of DNA identification. Forensic labs can store and match DNA records from convicted offenders and crime-scene evidence.

2. Steps taken before collection – notes, sketches, close-up photography

3. Precautions - disposable latex gloves; glasses or goggles; face masks; shoe covers; coveralls; very little personal contact!

4. Packaging – separately in a paper bag or well-ventilated box. Allow swabs to dry 5-10 minutes and place in a manila envelope. Place in refrigerator or cool place.

5. Substrate control – obtain a sample of the unstained surface with a swab

6. Control comparison – a. blood – obtain 7 cc of whole blood from victims and suspects for comparisonb. EDTA – a preservative for DNAc. DNA – swab the subject’s mouth

7. Contamination – introduction of foreign substances or foreign DNA into a sample. To prevent, always:

• Wear disposable gloves and a face mask

• Collect a substrate control• Pick up small items with clean forceps• Always package each item of evidence

in its own well-ventilated container.