2. DNA Extraction:
DNA can be extracted from
almost any human tissue.
•Buccal cells from inside cheek for
paternity tests.
•Sources of DNA at crime scene: blood,
semen, hair follicle, saliva.
•DNA extracted from evidence is
compared to DNA from known individuals
3. •Extracted DNA molecules are incubated
with restriction enzymes (endonucleases).
Restriction enzymes are produced by
bacteria as a defense against viruses.
These enzymes cut DNA at specific
base sequences called recognition
sites.
Results in smaller pieces of DNA
called RFLP’s.
5. RFLP Analysis:
•RF stands for Restriction Fragments. Those
are the fragments that were cut by restriction
enzymes.
•L stands for Length, and refers to the length
of the restriction fragment.
•P stands for Polymorphisms, a Greek term
for “many shapes”. The lengths of some of
the restriction fragments differ greatly
between individuals.
RFLP = Restriction Fragment Length
Polymorphism
6. Electrophoresis of these RFLP’s produce
different patterns of DNA bands.
With 3 billion base pairs in the human
genome, however, RFLP analysis would
produce a ‘smear’ of many similar sized
fragments.
Molecular biologists have identified regions
of the human genome where restriction
fragment lengths are highly variable
between individuals.
7. VNTR alleles are highly variable regions of
human DNA.
•VNTR stands for ‘variable number of
tandem repeats.
•A tandem repeat is a short sequence
of DNA that is repeated at a specific
chromosomal locus.
•Tandem repeats are interspersed
throughout the human genome.
8. VNTR’s continued:
•The number of repeats at a given
place on a certain chromosome is
highly variable from one person to
another.
•The number of such repeats is
usually different on the paternal and
maternal members of the same
person’s chromosome pair.
9. Red boxes represent the repeat unit and the blue lollipops
represent cut sites for a restriction endonuclease. (Here 3
different variants, may be 50 in reality).
10. •Analysis of a VNTR
locus most commonly
results in a two-band
pattern, one band
inherited from each
parent.
•A one-band pattern can
occur if the size of the
two parental bands are the
same or nearly the same.
•For our simple example of three different alleles
designated A, B, and C illustrated above, six unique
DNA profiles are possible.
12. RFLP Analysis:
•The RFLP markers most commonly used for DNA
profile analysis are found on chromosomes 1, 2, 4,
5, 10 and 17.
•These RFLP markers are named after their
locations on these chromosomes.
•
•For example, the marker on chromosome 2 is
called D2S44 (section 44 of chromosome 2).
•These chromosomal locations are also referred to
as DNA loci.
13.
14. •The Federal Bureau of Investigation (FBI) has
been a leader in developing DNA typing
technology for use in the identification of
perpetrators of violent crime.
•In 1997, the FBI announced the selection of 13
STR (short tandem repeat) loci to constitute the
core of the United States national database,
CODIS.
•All CODIS STRs are tetrameric repeat sequences.
•All forensic laboratories that use the CODIS
system can contribute to a national database.
15. •For example, D7S280 is one of the 13 core
CODIS STR genetic loci. This DNA is
found on human chromosome 7.
•The tetrameric repeat sequence of D7S280 is
"gata". Different alleles of this locus have
from 6 to 15 tandem repeats of the "gata"
sequence.
16. How many tetrameric repeats are present in the
DNA sequence shown below? Notice that one of the
tetrameric sequences is "gaca", rather than "gata".
1 aatttttgta ttttttttag agacggggtt tcaccatgtt
ggtcaggctg actatggagt
61 tattttaagg ttaatatata taaagggtat gatagaacac
ttgtcatagt ttagaacgaa
121 ctaacgatag atagatagat agatagatag atagatagat
agatagatag atagacagat
181 agatagtttt tttttatctc actaaatagt ctatagtaaa
catttaatta ccaatatttg
241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata
attcttaaga atatatattc
301 cctctgagtt tttgatacct cagattttaa ggcc
17. DNA profiles vary from person to person.
•When profiles from a single VNTR locus
from unrelated individuals are compared,
the profiles are normally different.
•However, it is possible for two individuals
to have the same profile at one or two loci.
•But the chance of more than one person
having the same DNA profile at 4, 5, or 6
different VNTR loci is extremely small.
18. DNA Profiling
DNA primers have been optimized to allow amplification
of multiple STR loci in a single reaction mixture.
19. Norma’s genotype is 15, 15 at the locus D3S1358, 14, 16
at vWA, and 24, 25 at FGA.
20. A DNA Profile: The 13 CODIS STR loci
Locus D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818
Geno- 15, 18 16, 16 19, 24 12, 13 29, 31 12, 13 11, 13
type
Fre- 8.2% 4.4% 1.7% 9.9% 2.3% 4.3% 13%
quency
Locus D13S317 D7S820 D16S539 THO1 TPOX CSF1P AMEL
O
Geno- 11, 11 10, 10 11, 11 9, 9.3 8, 8 11, 11 XY
type
Fre- 1.2% 6.3% 9.5% 9.6% 3.52% 7.2% Male
quency
21. How common or rare would this 13 locus DNA
profile be in the reference population?
In most cases, a "product rule" calculation can be
done by multiplying each individual probability
together
By combining the frequency information for all
13 CODIS loci, this frequency of this profile
would be 1 in 7.7 quadrillion Caucasians…that’s
1 in 7.7 x 1015 power!
