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Forensic Science
Presents
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 and
Guanine 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– electrophoresis
c. Digestion-by the restriction enzyme
d. Test gel
e. Prepare known samples
f. 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-G
e. multiplexing—using a variety of STR’s in order to further narrow
down the sample
1. importance—increases the probability of being only from
one source
2. 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 gene
1. chromosome location—sex chromosomes X
and Y
2. body location—tooth pulp
3. male—will show two bands
4. female—will show only one band
5. 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 mother
3. 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
comparison
b. EDTA – a preservative for DNA
c. 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.