advanced technologies in fs

8/3/2019 Advanced Technologies in FS

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ADVANCED DNA TECHNOLOGIES

IN FORENSIC SCIENCE

RANA MUHAMMAD ASIF

Forensic Scientist (DNA/Serology)

PUNJAB FORENSIC SCIENCE AGENCY

HOME DEPARTMENT

GOVERNMENT OF THE PUNJAB, PAKISTAN07/28/2010 1


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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

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Human Genome

Sequence Variation

Repeat Loci:

Micro-satellites (STRs) (


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

Forensic Genetics Timeline


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rensic Genetics:

seful Genetic Loci

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


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STR and VNTR Typing:

PCRAmplification ofRepeat Loci

Molecular Biology of the Cell, 4th Edition, pg511


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STR and VNTR Typing:

Mendelian Inheritance ofRepeat Loci

Progeny

Parents

Repeats


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Typing a Forensic Sample

Molecular Biology of the Cell, 4th Edition, pg511


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ensic STR Profile Comparison:

ManyLoci are Needed?

Nine Repeat Loci and Sex

What is a probability match and what does it mean?


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DNA TypingPortions of the DNA molecule containsequences of bases that are repeatednumerous times, known as tandemrepeats.To a forensic scientist, these tandemrepeats offer a means of distinguishing one

individual from another through DNAtyping.Tandem repeats seem to act as filler orspacers between the coding regions ofDNA.What is important to understand is that allhumans have the same type of repeats,but there is tremendous variation in thenumber of repeats each of us have.


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1. 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.

2. Typically, a core sequence consists of 15 to 35 bases in length and repeats

itself up to a thousand times.

3. The key to understanding DNA typing lies in the knowledge that numerous

possibilities exist for the number of times a particular sequence of base letterscan repeat itself on a DNA strand.

4. The presence or absence of certain recognition sites in a DNA sample

generates variable lengths of DNA fragments, which are separated using gel

electrophoresis.

5. They are then hybridized with DNA probes that bind to a complementary DNA

sequence in the sample.

6. RFLP is one of the original applications of DNA analysis to forensic

investigation.

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

Restriction Fragment Length Polymorphism (RFLP)


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RFLPOnce 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).


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A Positive RFLP Test Next, the nylon sheet is placed against X-ray

film and exposed for several days.

When the film is processed, bands appearwhere radioactive probes stuck to fragmentson the nylon sheet.

A typical DNA fragment pattern will show twobands (one RFLP from each chromosome).

When comparing the DNA fragment patternsof 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 differentprobes and combining their frequencies.


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PCR Testing Polymerase chain reaction is the outgrowth of

knowledge gained from an understanding ofhow DNA strands naturally replicate within acell.

For the forensic scientist, PCR offers a distinctadvantage in that it can amplify minutequantities 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 nucleotidesare added to rebuild each of the separatedstrands.

Now, this process is repeated 25 to 30 times.


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PCR and RFLP PCR technology cannot be applied to RFLP DNA

typing.

The RFLP strands are too long, often numbering in thethousands of bases.

PCR is best used with DNA strands that are no longerthan a couple of hundred bases.


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Short Tandem Repeats

The latest method of DNA typing,short tandem repeat (STR) analysis,has emerged as the most successful

and widely used DNA profilingprocedure.

STRs are locations on thechromosome that contain shortsequences that repeat themselveswithin the DNA molecule.

They serve as useful markers foridentification because they arefound in great abundancethroughout the human genome.


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STR Advantages STRs normally consist of repeating

sequences of 3 to 7 bases in length, and theentire strand of an STR is also very short,less than 450 bases in length.

This means that STRs are much lesssusceptible to degradation and may often berecovered from bodies or stains that havebeen subjected to extreme decomposition.

Also, because of their shortness, STRs areideal candidates for multiplication by PCR,thus overcoming the previously mentionedlimited-sample-size problem often associatedwith crime-scene evidence.


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The Power of STR

What makes STRs so attractive toforensic scientists is that hundredsof different types of STRs are found

in human genes. The more STRs one can

characterize, the smaller will be thepercentage of the population fromwhich a particular combination ofSTRs can emanate.

This gives rise to the concept ofmultiplexing.

Using the technology of PCR, onecan simultaneousl extract and


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Standardizing STR Testing

Currently, U.S. crime laboratorieshave standardized on 13 STRs forentry into a national database

(CODIS). A high degree of discrimination and

even individualization can beattained by analyzing a combinationof STRs (multiplexing) anddetermining the product of theirfrequencies.

With STR, as little as 125 picogramsof DNA is required for analysis.

This is 100 times less than that


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Y- STR Another tool available in the arsenal of theDNA analyst is the ability to type STRs

located on the Y chrosome, which is malespecific.

More than 20 different Y-STR markers havebeen identified.

Y-STRs will prove useful when multiple malesare involved in a sexual assault.

A Y-STR analysis will have only one band or

peak, rather than the conventional STR whichis derived from two chromosomes and hastwo bands or peaks.

The Y-STR is therefore less complicated inappearance and interpretation.


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Mitochondrial DNA Another type of DNA used for individual

characterization is mitochondrial DNA.

Mitochondrial DNA (mDNA) is located outside thecells 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.


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Mitochondrial DNA Testing Mitochondrial DNA typing does not approach

STR analysis in its discrimination power andthus 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 tonuclear DNA analysis.

Also, all individuals of the same maternallineage will be indistinguishable by mDNAanalysis.

Two regions of mDNA have been found to behighly variable and a procedure known assequencing is used to determine the order of

base pairs.


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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

19.4 DNA FINGERPRINTING


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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)



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VNTRsRestriction enzyme sites

DNA probes are used

to hybridize

specifically to the

repeat sequence

located within VNTRs


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TRADITIONAL

DNA

FINGERPRINTING


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The probe is called a

multilocus probe (MLS)

It binds to ~ 20 to 40

fragments of DNA that

contain the sequence


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RESTRICTION ENZYMES MECHANISM


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In the past decade, the technique of DNA

fingerprinting has become automated

It is now done using PCR, which amplifies short tandemrepeat 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 display19-67


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Restriction Enzymes Cut an Organisms DNA into

a Reproducible Set of Restriction Fragments


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GEL ELECTROPHORESIS


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Lane Sample1 DNA Ladder

2 Crime Scene

3 Suspect 1

4 Suspect 2

5 Suspect 3

Who committed the crime?

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Gel Electrophoresis Resolves DNA

Fragments of Different Size


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How are genomes of individuals different?

More than 90% of the differences take the form of substitutions at a single

base. These are called single nucleotide polymorphisms (SNP)

Nature 409, 822 - 823 (2001)

SNP

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FrequencyExampleRules for assigning allele to classClass of variation

5,692,700

(~93%)

A/TSingle base substitution involving

A,T,C, or G

Single Nucleotide

Polymorphism (SNP)

431,319

(~7%)

T/-CCTA/GDesignated using the full sequence of

the insertion as one allele, and either

a fully defined string for the variantallele or a - character to specify the

deleted allele.

Deletion/Insertion

Polymorphisms

(DIPs)

2,440

(0.04%)

(CAC)8/9/10/11Alleles are designated by providing

the repeat motif and the copy number

for each allele.

Microsatellite or

short tandem repeat

(STR)

1,859

(0.03%)

(alu) / -Applies to insertion/deletion

polymorphisms of longer sequence

features, such as retroposon

dimorphism for Alu or line elements.

Named variant

The Genetic Basis for Human Variation

Derived from dbSNP release 119

http://www.ncbi.nlm.nih.gov/SNP/07/28/2010

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Any 2 human genomes are roughly 99.9% identi

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

Chr - chromosome

n - Number of samples examined

bp - Number of basepairs sequences

S - Number of polymorphic sites

T - Nucleotide divergence

On average ~ 0.1%

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If 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)

Kruglyak and Nickerson Nature Genetics (2001) 27

Variation is the splice of life

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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 justa mutation )

How many SNPs are born in one generation?

Kruglyak and Nickerson Nature Genetics (2001) 27

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2,894,929

939,799

10,000,000

2,809,547,336

= 0.5%

13,834,728

(total genome)

Humans are 99.5% identical (not 99.9%)

S. Levy, PLoS Biol5 (2007), p. e25407/28/2010 42


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A locus is a region on the chromosomes

One locus

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An allele is a variation at a locus

allele frequency is the proportion

of a certain allele within a population.

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

CATTGCA..GTTTCTGATTTTTTGATGTCTTCATCCATCACTGTCCTTGTCAAATAGTTTCATTGCA..GTTTCTGATTTTTTGATGTCTTCAGCCATCACTGTCCTTGTCAAATAGTTT

For example in the same locus we may have:

llele 1:

Allele 2:

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What is the distribution of genotypes?

For two alleles ( and ) we have three genotypes:

Let p = the frequency of A , and

q = the frequency of a .

(p + q = 1)

What is the frequency of ?

AA Aa aaA a

= p2

= q2

= pq + qp = 2pq

AA Aa aa

AA

Aa

aa

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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 generation07/28/2010 46


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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

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

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POLYMORPHISM

Two kinds of polymorphic regions in the genome

Sequence polymorphismsLength 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

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Length Polymorphisms

Non-coding DNA has length polymorphismsParticular sequences may be repeated 1-30

times in variable number tandem repeats(VNTRs)

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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

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Isolate DNA from a sample (blood, saliva..)

Clean up the sample if retrieved from

clothing, carpeting, etc.

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Cut with restriction enzymes to produce

short, manageable DNA fragments

Bacterial enzymes used

DNA fragments range from 100 to 10,000base pairs in length

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PCR Analysis

PCR (polymerase chain reaction) is used to makemillions of exact copies of DNA from a biologicalsample.

DNA amplification with PCR allows DNA analysis onbiological samples as small as a few skin cells.

With RFLP, DNA samples would have to be about the size ofa quarter.

The ability of PCR to amplify such tiny quantities of

DNA enables even highly degraded samples to beanalyzed. Great care, however, must be taken to prevent

contamination with other biological materials duringthe identifying, collecting, and preserving of a sample.

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STR Analysis

Short tandem repeat (STR) technology is used toevaluate 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 missingpersons. The odds that two individuals will have the same 13-loci

DNA profile is about one in one billion.

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Y-Chromosome Analysis

The Y chromosome is passed directly from father toson, so the analysis of genetic markers on the Ychromosome is especially useful:

For tracing relationships among males or

For analyzing biological evidence involving multiple malecontributors.

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F t P t C i ti DNA E ti


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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 Americans77 Caucasians

21 Latinos

2 Asian American

4 whose race is unknown

Th t t d/ t t

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