DNA fingerprinting

1
DNA fingerprinting

• Goal
• Develop a unique identification tool to
  distinguish between people by using their DNA
• Applications
• Criminal identification and Forensics
• Paternity and Maternity
• Personal identification

2
Techniques to look at minisatellites

• Isolate DNA
• Digest and run on a gel, transfer to membrane
• Southern blotting with radioactive probe
• Multi locus probes
• Single locus probes
• Characterization of results

3
Multi locus probes

• Use for fingerprinting started in 1985
• Realization that although length of certain
  minisatellites was not polymorphic, using that
  sequence as a probe for low stringency Southern
  could detect other minisats that were polymorphic
• Found that using different minisatellites they
  could get unique banding patterns this way.
• Can detect around 36 bands with a single probe
• Problem- dont actually know which loci you are
  detecting, cant follow single alleles at those
  loci
• To maximize ability to see repeat sizes, remove
  as much flanking DNA as possible

4
VNTRs
DNA samples are cut with a restriction
endonuclease For forensic analysis,
HaeIII which cuts DNA at 5'-GGCC-3' sequences, is
used by many labs, including the FBI and Arizona
Department of Public Safety. Most
restriction fragments are the same from person to
person. Restriction fragments with VNTR
alleles (red bars) are highly variable from
person to person. The phenotype for
different alleles is a variable length
restriction fragment, which is detected by
agarose gel electrophoresis and Southern
hybridization. A known VNTR 17-bp in
length and repeated 70-450 times will have length
variation between 1.2 and 7.6 kbp.
5
Fingerprints

• A- paternity test
• F1, F2 possible fathers
• B- rape case
• Semen specimen found at scene of crime

6
Forensic applications
The image shows the test results in a rape case.
Two probes were used one revealing the bands
at the top, the other those at the bottom. DNA
was tested from - semen removed from the vagina
of the rape victim (EVIDENCE 2) - a semen stain
left on the victim's clothing (EVIDENCE
1) - the DNA of the victim herself (VICTIM) -
DNA from two suspects (SUSPECT 1, SUSPECT 2) -
a set of DNA fragments of known and decreasing
length (MARKER) -the cells of a
previously-tested person to be sure the
probes are performing properly (CONTROL)
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Single locus analysis- DNA profile

• Each probe detects two bands in any persons DNA
• Profiling relies on 4-10 different polymorphisms
• Can calculate exact probability if the gene
  frequency of each allele in a population is known
• Estimated that a pattern derived from 4
  locus-specific probes would be matched by chance
  less than once in a l million unrelated
  individuals
• Advantages
• Much more sensitive that multi-locus probes
  (require less DNA, 10 ng compared to 250 ng for
  multi locus probes)
• Measure both alleles of a population- allow you
  to measure allele frequencies in different ethnic
  populations (affect probability determination)

8
Six Different Phenotypes/Genotypes with only 3
VNTR Alleles DNAs from different
individuals can be distguished by analysis of
VNTR alleles. For a simple case of only 3
alleles, 3 heterozygous and 3 homozygous patterns
are possible (see diagram). For n
different alleles, there are (n)(n1)/2 possible
genotypes.
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Pedigree analysis

• chromosomes of the two parental individuals of
  the pedigree below. The first individual has one
  with 4 repeated sequences and one chromosome with
  6 repeated sequences. The other individual has
  one chromosome with 3 and one chromosome with 5
  repeated sequences.
• At the bottom of the figure is a pedigree of the
  mating between these two individuals and their
  four children. The DNA of each of the individuals
  has been analyzed for the VNTR repeat number and
  the gels are show below each individual along
  with the genotype for each individual. Notice
  that each of the six people are distinguishable
  from each other by the VNTR's at this one genetic
  locus. If several VNTR loci were used, the
  uniqueness of each individual would become even
  more distinct.

http//www.people.virginia.edu/rjh9u/vntr1.html
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STRs

• Starting in 1999, law enforcement agencies in
  both Great Britain and the United States began
  switching to a new version of RFLP analysis using
  shorter sequences called STRs ("Short Tandem
  Repeats").
• STRs are repeated sequences of a few (usually
  four) nucleotides, e.g., TCATTCATTCATTCAT. They
  often occur in the untranslated parts of known
  genes (whose sequence can be used for the PCR
  primers). The exact number of repeats (6, 7, 8,
  9, etc.) varies in different people (and, often,
  in the gene on each chromosome that is, people
  are often heterozygous for the marker).
• Using 10 different STRs - scattered over
  different chromosomes - the chance that two
  people picked at random have the same pattern is
  about 1 in 3 trillion. In the U.S., where 13 STRs
  are now routinely examined, the odds of a random
  match are even higher.

11
STR procedure

• Multiplex PCR amplifying 3-4 loci
• PCR products are labelled with fluorescent dyes
  and analysed using an automated DNA sequencer
• Advantages
• Tests are quick (2 hours)
• Very sensitive- require only about 0.1 ng DNA
• No need for radioisotopes or southern blotting
• Measurements of band migrations are very precise,
  allowing you to distinguish between closely
  migrating alleles
• It is tolerant of DNA degradation

12
VNTR Analysis of the Romanov Remains
-Tsar Nicholas, Tsarina Alexandra and their five
children were reported to have been assassinated
in the Russian Revolution along with the Royal
Physician and three servants. -An analysis of
the DNA from the bones was performed on DNA
amplified from five different short tandem
repeats (STR) of chromosomal DNA. -In each case,
the purported daughter has a STR genotype
consistent with the purported parents. STR
analysis of the remains of the physician and the
three servants indicated that the remains of
these four individuals were not those of members
of the Romanov family.
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Problems with DNA fingerprinting

• 1. Generating a High Probability
• The probability of a DNA fingerprint belonging to
  a specific person needs to be reasonably
  high--especially in criminal cases, where the
  association helps establish a suspect's guilt or
  innocence. Using certain rare VNTRs or
  combinations of VNTRs to create the VNTR pattern
  increases the probability that the two DNA
  samples do indeed match (as opposed to look
  alike, but not actually come from the same
  person)or correlate (in the case of parents and
  children).

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Problems with DNA fingerprinting

• 2. Problems with Determining Probability
• A. Population Genetics
• VNTRs are not distributed evenly across all of
  human population
• A given VNTR cannot,therefore, have a stable
  probability of occurrence it will vary depending
  on an individual's genetic background.
• The difference in probabilities is particularly
  visible across racial lines.
• Some VNTRs that occur very frequently among
  Hispanics will occur very rarely among Caucasians
  or African-Americans. Currently, not enough is
  known about the VNTR frequency distributions
  among ethnic groups to determine accurate
  probabilities for individuals within those
  groups the heterogeneous genetic composition of
  interracial individuals, who are growing in
  number, presents an entirely new set of
  questions.
• Further experimentation in this area of
  population genetics, has been surrounded with and
  hindered by controversy, because the idea of
  identifying people through genetic anomalies
  along racial lines could provide a scientific
  basis for racial discrimination.

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Probability

• Is suspect 1(slide 6) guilty? We can never be
  certain.
• The best we can do is to estimate the probability
  that another person, picked at random, could
  provide the same DNA fingerprint.
• As a conservative estimate, a given allele might
  be found in 25 of the people tested. The
  probability of a random match of two alleles is
  (0.25)2 or 1 in 16. The probability that 6
  alleles match, as in this case, is (0.25)6 or 1
  in 4096.
• But the suspect was not picked at random, so you
  may feel that the evidence of guilt is strong.
• The more probes you use, the more confident you
  can be that you have gotten the right man. If,
  for example, a set of probes revealed 14 bands in
  a suspect's DNA identical to those in the semen
  sample, the probability that you have the wrong
  man drops to less than 1 in 268 million (0.25)14
  1/268,435,456, which is more than the entire
  population, males and females, in the United
  States.
• http//users.rcn.com/jkimball.ma.ultranet/BiologyP
  ages/R/RFLPs.htmlfingerprinting

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Problems with DNA fingerprinting

• B. Technical Difficulties
• Errors in the hybridization and probing process
  must also be figured into the probability
• Most people will agree that an innocent person
  should not be sent to jail, a guilty person
  allowed to walk free, or a biological mother
  denied her legal right to custody of her
  children, simply because a lab technician did not
  conduct an experiment accurately.
• When the DNA sample available is minuscule, this
  is an important consideration, because there is
  not much room for error, especially if the
  analysis of the DNA sample involves amplification
  of the sample because if the wrong DNA is
  amplified (i.e. a skin cell from the lab
  technician) the consequences can be profoundly
  detrimental.
• Until recently, the standards for determining DNA
  fingerprinting matches, and for laboratory
  security and accuracy which would minimize error,
  were neither stringent nor universally codified,
  causing a great deal of public outcry.

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Description of Services Establishment of
paternity and maternity Identification
of mutilated dead bodies Establishment
of biological relationships for immigration,
organ transplantation and property inheritance
matters Identification of missing
children and child swapping in hospitals
Identification of rapist in rape cases
Identification in murder case Species
identification in wildlife poaching cases New
services proposed Certification of meat
and other biological food products using
state-of-art DNA technologies
Monitoring of GM foods Pedigree
determination of Kennel and domestic pets
Identification of endangered species and
prevention of wildlife poaching Databank
services for criminal justice administration
Fingerprinting of plants and seeds for patent
purposes
http//www.cdfd.org.in/gvrser.html
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That brings me to genetic manipulation of animals

• Two major research areas have benefited
• Studies of gene function
• Animal models of disease
• Need to genetically modify germline of animals
• Fertilized oocyte (creates fuly transgenic
  animal)
• Embryonic Stem cells (ES) (creates mosaic)
• Any animal with a foreign DNA molecule inserted
  in it is called transgenic

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Pronuclear microinjection to create transgenic
mouse
Inject into male pronucleus- its larger Usually
see insertion of multiple copies Technically
difficult
20
ES cells

• ES cells removed from 3.5-4.5 day old embryos,
  arise from inner cell mass of blastocyst
• Can be cultured in vitro and retain potential to
  contribute to all tissues, including germline
• Get injected back into a host blastocyst and
  reimplanted in a pseudopregnant mouse
• Developing embryo is a chimera
• Contains cells from implanted ES cells and from
  host blastocyst
• Use strains with different coat colours- can
  easily distinguish between them
• ES cells may form some of germline, select for
  offspring carrying correct coat colour

21
Genetically modified ES cells as a route for
transferring foreign DNA or specific mutations
into the mouse germline
22
Gene targeting by homologous recombination
Homologous recombination can inactivate a
predetermined chromosomal gene within an intact
cell A. insertion vector method B. replacement
vector method
23
Double replacement gene targeting
-To introduce subtle mutations -Since methods on
last slide result in incorporation of exogenous
sequence - uses positive-negative
selection Note- these mice are not transgenic
24
Knock in-method of gene replacement
25
Site-specific recombination systems

• Cre-loxP system from bacteriophage P1
• Cre recombinase mediates recombination between
  two loxP sequences that are in the same
  orientation
• As a result of recombination, the sequences
  between the loxP sites is deleted

26
Gene targeting using Cre-loxP Step
one -Introduce 3 loxP sites along with a marker
M by homologous recombination in ES cells -
transfection and expression of Cre recombinase
generates different products - Type 1
recombinants used to make transgenic mice
27
Gene targeting using Cre-loxP Step two - Mate
mouse from previous slide with mouse carrying
integrated Cre recombinase gene linked to a
tissue specific promoter - offspring which
contain both loxP-flanked target locus and Cre
will express Cre in desired tissue and induce
recombination, resulting in tissue specific
inactivation of target gene A
28
Chromosomal engineering to generate
translocations and deletions