Forensics

14.2.19 BCMB 612

 DNA forensics
• DNA fingerprinting useful to identify crime
suspects from biological evidence collected at
the crime scene.

• Biological samples for DNA fingerprinting include

blood, semen, hair, skin, urine, saliva, bone,
teeth, tissues, etc.

• Samples must contain undegraded DNA

 Contact and Appointments
DNA Center Ghana
7th floor, GNAT Heights
30 Independence Avenue
Ridge, Accra

Phone
030 293 2267
DNA Centre Ghana
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
 Forensic role of DNA profiling

• identify potential suspects whose DNA may match

evidence left at crime scenes

• exonerate persons wrongly accused of crimes

• identify crime and catastrophe victims

• establish paternity and other family relationships

 Performance of Forensic Tools

http://www.bio-rad.com/
 Blood typing - limitations
Blood types (Groups A, B, AB, O, Rhesus):

Low specificity since 40% pop blood type O.

Useful to exclude suspect(s) whose blood

type not found at crime scene.
Restriction Fragment Length Polymorphism
(RFLP)

– RFLP target DNA large repeat tandem sequence

– (VNTR) variable number tandem repeats

– discovered by Sir Alec Jeffries (1985)

– Number of repeats vary with individuals;

– restriction enzyme digest at specific chromosomal sites

used to define length of repeat units.
Microsatellite 2-4 bp repeats
Mini satellite (larger 20-50 bp repeats)
 STR
• Microsatellites 2-6 bp repeats
• Number of STR repeats highly variable
among individuals - desired for ID
purposes
• STR found as introns, non-coding regions -
3% of total genome
• Easily amplified by PCR using primers
designed to target invariant flanking
regions
 STR
Repeat unit alleles No of alleles

Mononucleotide A, C, G, T 4

Dinuleotide AC, AG, AT, CG............. 16

Trinucleotide AAC, AAG, AAT, ACC..... 64

Tetranucleotide AAAC, AAAG, AAAT........ 256

Pentanucleotide AAAAC, AAAAG............. 1024

Hexanucleotide AAAAAC, AAAAAG....... 4096

 STR: HUMTH01
TH01 occurs as intron human tyrosine dehydrogenase gene

The TH01 locus contains repeats of TCAT.

CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA

This example has 6 TCAT repeats.

There are more than 20 known TH01 alleles.

Each individual inherits 1 allele from each parent.

http://www.bio-rad.com/
 STR

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
 Mini-satellite DNA fingerprinting

• Mini-satellites discovered by Sir Alec Jeffreys (1985)

• Variable number tandem repeat sequences (VNTRs)
• Unique sequences (20-50 base pairs long)

• repeat units range 50 to several hundred-fold

• occur in the non-coding regions of DNA (introns)

• number of tandem repeat units at specific chromosome sites vary
between individuals;
• Variability due to loss of tandem repeats during DNA replication
• Unrelated individuals have mini-satellites of different lengths
• Children inherent one set of mini-satellite sequence from each parent
• Mini-satellite pattern (DNA fingerprint) specific for each individual
• Probability of 2 individuals with the same fingerprint less than 1%
 DNA fingerprinting
2 approaches:
Classical RFLP: DNA hybridization to minisatellites, VNTR
(require undegraded DNA samples)

PCR amplification and DNA hybridization to microsatellites, STR

(highly sensitive, can use minute or degraded DNA samples)
 RFLP vs. PCR

Feature RFLP PCR

Time required Long (southern blot) Rapid (real time)

Intermediate (time
saver RE & gel run)
Amt of DNA 50-500 ng 0.1-1 ng

Quality of DNA Intact DNA Degraded DNA

Form DNA Double-stranded Single/double-

stranded
Discrimination 1 in 1 billion 1 in 1 billion
(6 loci) (8-13 loci)
 Advantages of PCR
• Need very small amts of DNA sample
• Can use degraded DNA (few 100 bps)
• Amplify multiple loci simultaneously
Mutiplex PCR
• Use human-specific primers to avoid
microbial DNA contamination
• Problems PCR inhibitors: hemoglobin
(blood); melanin (hair); indigo (jeans)
PCR Forensic test

www.accessexcellence.org
PCR-assisted Forensic tests

www.accessexcellence.org
 RFLP Markers
Not sex linked, males and female are equally likely to inherit either
of parental alleles

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
 Classical RFLP (southern blot) procedure

1. Extract and purify DNA from sample

2. Treat DNA sample with restriction enzyme (cuts specific

sites near VNTR)

3. Gel electrophoresis of restriction DNA fragments

4. Southern transfer of DNA fragments band to nylon

membrane; (strong alkaline to give single stranded DNA)

5. Add radioactive-labeled DNA probe with complementary

sequence to hybridize VNTR bands on nylon membrane

6. Wash unbound DNA probe and expose membrane to

X-ray film to develop restriction fragments bands
Southern blotting
 Immigration case
Sarbah vs. UK Home Office (1983)
• 13-yr-old Ghanaian child (Andrew) denied entry to UK to
join mother (Christiana Sarbah) and other siblings
resident in UK.

• First application of Alec Jeffrey’s DNA profiling technique

to prove that Andrew was truly the biological son of
Christiana.

• Read case details:

http://dnafingerprinting19.tripod.com/id14.html

http://www.dnalc.org/ddnalc/resources/shockwave/dnadetective.html
RFLP Forensic Anaylsis

http://www.bio-rad.com/
Forensic Analysis

http://www.bio-rad.com/
 Somatic vs Sperm DNA
• Somatic DNA tightly coiled surrounded by basic
histone proteins to neutralize repulsive –ve charges
in DNA.
• Sperm DNA specialized packing surrounded by
cysteine-rich protamine proteins containing
disulfide bridges
• Sperm DNA resistant to detergents and proteolytic
attack unless disulfide bonds reduced
• Can separate semen DNA from vaginal tissue DNA
in rape cases
Rape case

http://www.genelex.com/
 DNA Extraction Protocol
• Homogenize tissue in alkaline solution containing EDTA
(chelate Mg++ inhibit Dnases); add spermine (+ve charges to
stabilize DNA)
• Add anionic detergents (SDS) and proteases (proteinase K) to
solubilize and digest histones
• Extract into organic solvent phenol-chloroform to inactive
proteases
• Precipitate DNA with alcohol-salt solution
• Sperm DNA requires treatment with thiol reducing agents (DTT)
to reduce disulfide bridges in protamine (resistant to
detergents)
• Minimize problems with microbial contamination and shear
damage
 CODIS Chromosomal Loci
Combined DNA Index System – US FBI database
Chromosomal loci of STRs

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
 SGM (Second Generation Multiplex)
Locus Chromosome Common Allele Size Range
Dye Label
Designation Location Sequence Motif range (bp)
(TTTC)3TTTT
FGA 4q28 TTCT (CTTT)n 12.2-51.2 215–353 NED
CTCC (TTCC)2
TH01 11p15.5 (AATG)n 3-14 165–204 NED
TCTA(TCTG)3-
VWA 12p12-pter 10-25 157–209 5-FAM
4(TCTA)n

D2S1338 2q35–37.1 (TGCC)n(TTCC)n 15-28 289–341 5-FAM

TCTA (TCTG)1-3
D3S1358 3p 8-21 114–142 5-FAM
(TCTA)n
D8S1179 8 (TCTR)n 7-20 128–172 JOE
D16S539 16q24-qter (AGAT)n 5-16 234–274 5-FAM
D18S51 18q21.3 (AGAA)n 7-39.2 26–345 JOE
(AAGG)(AAAG)
D19S433 19q12–13.1 (AAGG)(TAGG) 9-17.2 106–140 NED
(AAGG)n

(TCTA)n(TCTG)n[(
TCTA)3TA(TCTA)3
D21S11 21q11.2–q21 TCA 12-41.2 187–243 JOE
(TCTA)2TCCA TA]
(TCTA)n

X: p22.1–22.3
Amelogenin — — 107 113 JOE
Y: p11.2
CODIS
STR Locus Evidence Suspect A Suspect B Suspect B's Genotype
Sample Frequency for Each STR

D3S1358 15, 17 17, 17 15, 17 0.13

vWA 15, 16 18, 19 15, 16 0.22

FGA 23, 27 21, 23 23, 27 0.31
D8S1179 12, 13 14, 15 12, 13 0.34

D21S11 28, 30 27, 30.2 28, 30 0.06

D18S51 12, 18 14, 18 12, 18 0.11

D5S818 13, 13 9, 12 13, 13 0.29

D13S317 12, 12 12, 12 12, 12 0.21

D7S820 10, 11 9, 10 10, 11 0.26

CSF1PO 8, 11 11, 12 8, 11 0.18

TPOX 7, 8 8, 8 7, 8 0.30
THO1 9.3, 9.3 6, 9.3
9.3, 9.3 0.38
D16S539 9, 13 11, 12
9, 13 0.10
 PARTIAL MATCHES
(1) A partial match is not an exact profile match of the two
profiles and may be due to:
(1) Partially degraded DNA
(2) DNA from crime scene containing a mix from different people
(3) A close relative of the suspect may be the true suspect
(4) A suspect can not be convicted based on moderate stringency
results

(2) true siblings will very rarely share alleles at all CODIS core loci;

(3) as offender DNA databases increases, the number of

unrelated people that share at least one allele at all loci
increases very rapidly.
 ABI 3500 Genetic Analyzer
8 channel capillary electrophoresis separate DNA fragments
by size
Fluorescence dye-labelled DNA fragments detected by laser
Fluorescent-labeled PCR
 Barcode
(DNA Fingerprint)
Analysis of multiple STRs

http://www.genelex.com/paternitytestin
 Which tests to use:
• For missing person, relatives of missing person, and
unidentified human (remains) samples
– all CODIS Core Loci must be attempted.
– additional DNA technologies (such as mtDNA, Y STR, Amelogenin)
should always be considered, as appropriate.

• For a crime scene investigation

– all CODIS Core Loci must be attempted but at least 8 of the original
CODIS Core Loci combined with a match rarity of at least one in ten
million are required for submission to and searching at NDIS.
– For Missing Person and Unidentified Human Remains,

• For paternity testing

– all CODIS Core Loci must be attempted but at least 8 of the original
CODIS Core Loci to include Y STR and Amelogenin
Mitochondrial
DNA
 mtDNA
• mtDNA available in high copy nos per cell
used where sample material is limited

• Fairly stable over generations - useful to

trace ethnicity

• Maternal relatives inherit same mtDNA - less

discriminating for individual identification
 Mitochondrial DNA

• Circular DNA consisting of 16,600 bps

Each mitochondria has 5-10mt DNA
• Each cell has 100-1000 mitochondria (high copy number)
• mtDNA inherited from mother during reproduction (sperm
contributes only nuclear DNA)
• mtDNA sequence provide unbroken chain of maternal
inheritance “ancestral EVE”
• High rate of mutations in certain hyper-variable (non-coding)
regions (lack DNA repair system)
• Sequence analysis of markers in hyper-variable mtDNA
regions useful to trace genealogy and ancestry along the
female line among communities
• STR sequence HVR-1 (16024-16365) & HVR-2 (73-340)
http://www.kerchner.com/mtdnachart.htm
mt DNA

http://users.rcn.com/jkimball.ma.ultranet/Biology
 Y-chromosome
• Sex-linked chromosomes:XX (Female) vs. XY
(Male); Y-chromosome inherited along male lines

• Not subject to genetic crossover & recombination

during reproduction; highly conserved sequences

• Consists 50 million bps; 220 genes

• Mutations used as markers to trace paternal

lineage

• Haplogroups Genghis Khan

Y chromosome
markers

12 loci from the non-recombining

region of the Y- chromosome:

DYS 19, DYS 385a/b, DYS389i,

DYS389ii, DYS390, DYS391,
DYS392, DYS393, DYS437,
DYS438, DYS 439 and DYS287.
Historical evidence : Y-chromosome lineage showed that US
President Thomas Jefferson fathered children with slave mistress
(Sally Hemings)

http://www.kerchner.com/mtdnachart.htm
 African Ancestry Project
• Genetic link to trace ‘roots’ of descendants
Africans transplanted to the diaspora during the
slave trade

• Analysis of mt DNA and Y-DNA for genetic

markers with reference to current African tribes

• Database of mtDNA and Y-DNA maps of African

sub-populations
Ancestry: Human Genealogy