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RANA MUHAMMAD ASIF Forensic Scientist (DNA/Serology)



DNA Technologies in Forensic Investigations

Restriction Fragment Length Polymorphism (RFLP)

Polymerase Chain Reaction (PCR) Analysis Short tandem repeat (STR) technology Mitochondrial DNA Analysis Y-Chromosome Analysis


Human Genome Sequence Variation

Repeat Loci: Micro-satellites (STRs) (<10bp, CGn) Mini-satellites (VNTRs) (10bp< length <100bp) Sequence Polymorphisms: Coding and Non-Coding Point Mutations (SNPs) Structural Variation Duplications, Inversions, Deletions

Allelic Frequency vs Genotypic Frequency

Forensic Genetics Timeline

Jobling and Gill (2004) Nature Reviews Genetics 5:739-751

Forensic Genetics: Useful Genetic Loci

Jobling and Gill (2004) Nature Reviews Genetics 5:739-751

STR and VNTR Typing: PCR Amplification of Repeat Loci

Molecular Biology of the Cell, 4th Edition, pg511

STR and VNTR Typing: Mendelian Inheritance of Repeat Loci

Repeats Parents


Typing a Forensic Sample

Molecular Biology of the Cell, 4th Edition, pg511

orensic STR Profile Comparison: ow Many Loci are Needed?

Nine Repeat Loci and Sex

What is a probability match and what does it mean?

DNA Typing
Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats. To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA typing. Tandem repeats seem to act as filler or spacers between the coding regions of DNA. What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have.


Restriction Fragment Length


Polymorphism (RFLP)

RFLP analyzes the variable lengths of DNA fragments that result from digesting a DNA sample with a special restriction endonuclease that cuts DNA at a specific recognition site. Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times. The key to understanding DNA typing lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample. RFLP is one of the original applications of DNA analysis to forensic investigation.








It requires relatively large amounts of DNA and do not work well degraded samples

Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis. The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones. The fragments are then transferred to a nylon membrane in a process called Southern blotting. To visualize the RFLPs, the nylon sheet is treated with radioactive probes containing a base sequence

complementary to the RFLPs being identified (a process called hybridization).


Next, the nylon sheet is placed against X-ray film and exposed for several days. When the film is processed, bands appear where radioactive probes stuck to fragments on the nylon sheet. A typical DNA fragment pattern will show two bands (one RFLP from each chromosome). When comparing the DNA fragment patterns of two or more specimens, one merely looks for a match between the band sets. A high degree of discrimination can be achieved by using a number of different probes and combining their frequencies.

A Positive RFLP Test

Polymerase chain reaction is the outgrowth of knowledge gained from an understanding of how DNA strands naturally replicate within a cell. For the forensic scientist, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times. First, the DNA is heated to separate it. Second, primers (short strands of DNA used to target specific regions of DNA for replication) are added which hybridize with the strands. Third, DNA polymerase and free nucleotides are added to rebuild each of the separated strands. 14 Now, this process is repeated 25 to 30 times.

PCR Testing

PCR technology cannot be applied to RFLP DNA typing. The RFLP strands are too long, often numbering in the thousands of bases. PCR is best used with DNA strands that are no longer than a couple of hundred bases.



One advantage in moving to shorter DNA strands is that they would be expected to be more stable and less subject to degradation brought about by adverse environmental conditions. The long RFLP strands tend to readily break apart under the adverse conditions not uncommon at crime scenes. PCR also offers the advantage in that it can amplify minute quantities of DNA, thus overcoming the limited sample size problem often associated with crime scene evidence.

PCR Advantages

Short Tandem Repeats

The latest method of DNA typing, short tandem repeat (STR) analysis, has emerged as the most successful and widely used DNA profiling procedure. STRs are locations on the chromosome that contain short sequences that repeat themselves within the DNA molecule. They serve as useful markers for identification because they are found in great abundance throughout the human genome.



STRs normally consist of repeating sequences of 3 to 7 bases in length, and the entire strand of an STR is also very short, less than 450 bases in length. This means that STRs are much less susceptible to degradation and may often be recovered from bodies or stains that have been subjected to extreme decomposition. Also, because of their shortness, STRs are ideal candidates for multiplication by PCR, thus overcoming the previously mentioned limited-sample-size problem often associated with crime-scene evidence.

STR Advantages

The Power of STR

What makes STRs so attractive to forensic scientists is that hundreds of different types of STRs are found in human genes. The more STRs one can characterize, the smaller will be the percentage of the population from which a particular combination of STRs can emanate. This gives rise to the concept of multiplexing. Using the technology of PCR, one can simultaneously extract and


Standardizing STR Testing

Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS). A high degree of discrimination and even individualization can be attained by analyzing a combination of STRs (multiplexing) and determining the product of their frequencies. With STR, as little as 125 picograms of DNA is required for analysis. This is 100 times less than that


Another tool available in the arsenal of the DNA analyst is the ability to type STRs located on the Y chrosome, which is male specific. More than 20 different Y-STR markers have been identified. Y-STRs will prove useful when multiple males are involved in a sexual assault. A Y-STR analysis will have only one band or peak, rather than the conventional STR which is derived from two chromosomes and has two bands or peaks. The Y-STR is therefore less complicated in appearance and interpretation. 21


Another type of DNA used for individual characterization is mitochondrial DNA. Mitochondrial DNA (mDNA) is located outside the cells nucleus and is inherited from the mother. Mitochondria are structures found in all our cells used to provide energy that our bodies need to function. A single mitochondria contains several loops of DNA.

Mitochondrial DNA


Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best reserved for samples, such as hair, for which STR analysis may not be possible. Forensic analysis of mDNA is more rigorous, time consuming, and costly when compared to nuclear DNA analysis. Also, all individuals of the same maternal lineage will be indistinguishable by mDNA analysis. Two regions of mDNA have been found to be highly variable and a procedure known as sequencing is used to determine the order of 23 base pairs.

Mitochondrial DNA Testing


DNA fingerprinting is a technology that identifies particular individuals using properties of their DNA
It is also termed DNA profiling

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

When subjected to DNA fingerprinting, chromosomal DNA gives rise to a series of bands on a gel
The order of bands is an individuals DNA fingerprint It is the unique pattern of these bands that makes it possible to distinguish individuals

Certain loci in human chromosomes are variable in length

These loci contain tandemly repeated sequences called minisatellites Variable Number of Tandem Repeats (VNTRs)

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Restriction enzyme sites


DNA probes are used to hybridize specifically to the repeat sequence located within VNTRs

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display


The probe is called a multilocus probe (MLS) It binds to ~ 20 to 40 fragments of DNA that contain the sequence






In the past decade, the technique of DNA fingerprinting has become automated
It is now done using PCR, which amplifies short tandem repeat sequences (STRs) Like VNTRs, STRs are found in multiple sites in human genomes and are variable among different individuals The main difference between a VNTR and STR is size
STRs are much shorter, usually 100450 bp STRs are called microsatellites, and VNTRs minisatellites

The amplified STRs are fluorescently labeled They are separated by gel electrophoresis A laser excites the fluorescent molecule within the STR A detector records the amount of emission for each STR
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display


Restriction Enzymes Cut an Organisms DNA into a Reproducible Set of Restriction Fragments






Gel Electrophoresis Resolves DNA Fragments of Different Size

Lane Sample 1 DNA Ladder 2 Crime Scene 3 Suspect 1 4 Suspect 2 5 Suspect 3

Who committed the crime?

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Human Genetic Variation

With the exception of monozygotic twins, every one of us is genetically different from every other human who ever lived.

American Express 1990 Advertisement 07/28/2010 36 http://www.childrenofsalem.com/days/kids/ericbran/ericbran1.html

How are genomes of individuals different?


Nature 409, 822 - 823 (2001) 07/28/2010

More than 90% of the differences take the form of substitutions at a single 37 base. These are called single nucleotide polymorphisms (SNP)

The Genetic Basis for Human Variation

Class of variation Single Nucleotide Polymorphism (SNP) Deletion/Insertion Polymorphisms (DIPs)

Rules for assigning allele to class Single base substitution involving A,T,C, or G Designated using the full sequence of the insertion as one allele, and either a fully defined string for the variant allele or a - character to specify the deleted allele. Alleles are designated by providing the repeat motif and the copy number for each allele. Applies to insertion/deletion polymorphisms of longer sequence features, such as retroposon .dimorphism for Alu or line elements

Example A/T

Frequency 5,692,700 (~93%) 431,319 (~7%)


Microsatellite or short tandem repeat (STR) Named variant


2,440 (0.04%)

(alu) / -

1,859 (0.03%)


Derived from dbSNP release 119 http://www.ncbi.nlm.nih.gov/SNP/


Any 2 human genomes are roughly 99.9% identica

Chr - chromosome n - Number of samples examined bp - Number of basepairs sequences S - Number of polymorphic sites 07/28/2010 T - Nucleotide divergence

On average ~ 0.1%

39 Przeworski, M., et al. (2000) Trends Genet 16, 296-30

Variation is the splice of life


1. Any two genomes are roughly 99.9% identical and 2. A genome is 3.2 billion base pairs long

Then: Every two genomes have 3.2 million differences (SNPs) (remember also that each individual has two genomes)


40 Kruglyak and Nickerson Nature Genetics (2001) 27 2

How many SNPs are born in one generation?

Number of genomes: N = 14*109 (twice the number of people, why?) Mutation rate: Q = 2*10-8 per base-pair per generation (gen) New mutations = NQ = 280 per base-pair per generation In other words: Every base gets mutated in 280 individuals each gen However the overwhelming majority of these will be very rare. We set a minimal frequency for a polymorphism at 1% in the population (below this frequency its just a mutation )


41 Kruglyak and Nickerson Nature Genetics (2001) 27 2

Humans are 99.5% identical (not 99.9%)



10,000,000 (total genome) 2,809,547,336 = 0.5%

13,834,728 S. Levy, PLoS Biol 5 (2007), p. e254



A locus is a region on the chromosomes

One locus



An allele is a variation at a locus For example in the same locus we may have:


allele frequency is the proportion of a certain allele within a population.

07/28/2010 http://www.micro.utexas.edu/courses/levin/bio304/popgen/popgen.html


What is the distribution of genotypes?

For two alleles (

and A

) we have three genotypes: a , and .




Let p = the frequency of A q = the frequency of a (p + q = 1) What is the frequency of



aa = p2

AA = q2 aa

= pq + qp = 2pq


The Hardy-Weinberg equilibrium


p2 + 2pq + q2 = 1 (p + q)2 = 1 p = (p2 + pq)/(p2 + 2pq + q2) q = (q2 + pq)/(p2 + 2pq + q2) p is the frequency of p in the next generation


Hardy-Weinberg equilibrium Assumptions of the standard random-mating model random mating no mutation no migration no selection the population size is infinitely large

Frequencies for some alleles can be very close to the equilibrium values

07/28/2010 http://www.micro.utexas.edu/courses/levin/bio304/popgen/popgen.html


Two kinds of polymorphic regions in the genome
Sequence polymorphisms Length polymorphisms Sequence Polymorphisms
Usually simple substitutions of one or two bases in the genes themselves. Genes comprise only 5% of genome. Non-coding DNA is other 95% Regulates gene expression Aids or impedes cellular machinery Chromosomal structure



Length Polymorphisms

Non-coding DNA has length polymorphisms Particular sequences may be repeated 1-30 times in variable number tandem repeats (VNTRs)



Creating a DNA Profile

Restriction Fragment Length Polymorphism analysis determines the number of VNTR repeats at a distinctive loci Count the number of repeats within the VNTR area



Creating a DNA Profile

Isolate DNA from a sample (blood, saliva..) Clean up the sample if retrieved from clothing, carpeting, etc.



Creating a DNA Profile

Cut with restriction enzymes to produce short, manageable DNA fragments Bacterial enzymes used DNA fragments range from 100 to 10,000 base pairs in length



PCR Analysis
PCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells.
With RFLP, DNA samples would have to be about the size of a quarter.

The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.
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STR Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in one billion.
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Analysis of Mitochondrial DNA

Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA.
In contrast to nuclear DNA, mtDNA analysis uses DNA extracted from mitochondrion.

In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable. All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing person investigations.
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Y-Chromosome Analysis
The Y chromosome is passed directly from father to son, so the analysis of genetic markers on the Y chromosome is especially useful:
For tracing relationships among males or For analyzing biological evidence involving multiple male contributors.



Facts on Post-Conviction DNA Exonerations

There have been 255 post-conviction DNA exonerations in the United States. The first DNA exoneration took place in 1989. Exonerations have been won in 34 states; since 2000, there have been 189 exonerations. 17 of the 255 people exonerated through DNA served time on death row. The average length of time served by exonerees is 13 years. The total number of years served is approximately 3,245. The average age of exonerees at the time of their wrongful convictions was 27. Races of the 255 exonerees: 151 African Americans 77 Caucasians 21 Latinos 2 Asian American 4 whose race is unknown
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The true suspects and/or perpetrators have been identified in 111 of the DNA