DNA

1
DNA

• Forensic Applications
• By Christ Abikaram
• SBF6-02
• Instructor- Ms. Villani

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What is DNA?

• DNA is known as Deoxyribonucleic Acid
• Consists of a nitrogenous base, phosphate group,
  and fiver carbon sugar (deoxiribose)

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Where can DNA be found in the human body?

• DNA is double stranded and forms hydrogen bonds
  between complementary nitrogenous bases
• This double stranded DNA is then coiled around
  histones to create compaction
• This is then even further compacted to form
  chromatin fibers
• Furthermore, they are highly compacted into
  chromosome
• Found in the nucleus of somatic and germ cells
• Need to be removed from nucleus for forensic
  analysis

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Where can DNA be found in the crime scene?

• Blood
• Sweat
• Hair Roots
• Saliva
• Various Tissue
• Semen

5
Types of physical evidence that may have DNA

• Chewing Gum
• Stamps Envelopes
• Penile Swabs
• Washed Stains
• Door Knobs
• Tooth Brushes
• Sanitary Pads

• Tooth Pulp
• Sweaty Clothing
• Telephones
• Bone Marrow
• Hair Brushes

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How do we isolate DNA?

• First we must break open these cells, which may
  be done in two ways by grinding or through
  ultrasonic sound to disrupt the particles
  maintaining the cell
• Second, we remove the phospholipid-bilayer
  containing lipids and fats by using a detergent
• Thirdly, we use chemicals such as protease to
  remove histone proteins that are still bound to
  the coiled chromosomes and degrade other
  proteins, leaving us with simple DNA
• This DNA can be resolubilized through washing
  with a wash buffer as well as with an alkaline
  buffer

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How do we amplify this extracted DNA?

• Before any experimentation can be done, it is
  necessary to amplify this DNA through a process
  known as PCR
• Through this process, as seen in the diagram to
  the right, we end up with multiple copies of the
  DNA in which we can now perform specific tests
  with

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What is PCR?

• Polymerase chain reaction (PCR) is a four step
  process used in genetic engineering to replicate
  as many copies of your DNA genome as possible
• There are three steps to this process
  denaturation, annealing, and elongation

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How is PCR completed?

• Through denaturation we heat the DNA to create
  two separate single strands in a solution with
  heat-capable DNA polymerase and primers and other
  nucleotides
• During annealing we cool down the solution
  allowing the primers to hydrogen bond with the
  ends of the target sequence
• During the extension period, DNA polymerase
  continues with the nucleotide sequence by adding
  to the 3 prime end of each primer
• Repeating this in many cycles helps create
  multiple copies of identical DNA which can than
  be used for further experimentation and forensic
  analysis

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Significance of PCR

• Now that there are many copies of the selected
  DNA, forensic analysis and tests can take place
• Gel electrophoresis and Southern Blotting can
  help in DNA fingerprinting and comparison between
  victim and suspect blood
• Mitochondrial DNA from forensic entomology found
  in insects can be magnified and help identify the
  deceased
• Paternity and family relationships can be
  determined using RFLP analysis

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Gel Electrophoresis

• Different DNA strands can be placed in the wells,
  and through an electric current, the negatively
  charged DNA will move based on its different size
• Because most human DNA genomes are incredibly
  long and repetitive, RFLP analysis must be
  completed before Gel Electrophoresis
• Otherwise, the strands of DNA will all be
  scattered and blurry because of their very
  similar lengths
• Gel Electrophoresis separates each strand due to
  its size because larger particles are not able to
  travel through a specific type of gel

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RFLP Analysis

• Due to DNAs longevity and repetition,
  restriction enzymes are necessary in order to
  create clear distinct fragment lengths
• Because each and every genome of a human being is
  original (minus that of identical twins) RFLP has
  an incredibly small chance of having the same
  length fragments

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Purpose of RFLP

• Restriction Fragment Length Polymorphism is
  incredibly important in forensic cases
• It allows a simple way to extract and analyze DNA
  and come up with matches between DNA samples from
  the victim, suspect, and on physical evidence

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Further Purposes of RFLP

• If blood is found on a shirt and bed, RFLP
  analysis on the DNA will determine whether this
  blood had come from the same victim
• This is completed by comparing the matching bands
  created by the restriction enzymes used
• Same bands indicate same DNA and therefore
  created a correlation between the victim and
  physical evidence
• This can also be compared with the victims DNA
  or suspects to see whose blood this belonged to

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Southern Blotting

• Along with RFLP analysis, 5 restriction sites are
  commonly used from criminal cases, Southern
  Blotting also helps make clear distinctions
  between various DNA samples
• With the gel arranged above a sponge between an
  alkaline solution and the blot paper
  (nitrocellulose), capillary action pulls the
  alkaline solution upward through the gel,
  transferring the DNA to the blot

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Nucleic Acid Hybridization

• After blotting the DNA onto the blot paper, the
  next step is nucleic acid hybridization
• The paper blot is exposed to a solution
  containing a radioactive labeled probe
• This probe will bind to the fragments leave a
  radioactive trace
• This radioactive probe can be detected through
  autoradiography using a photographic film sheet,
  as seen in this image to the right

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Importance of Southern Blotting

• The nucleic acid probe is a sequence of DNA that
  is contained within a library of genomes
• The results of the autoradiography will be those
  containing the specific DNA fragments of the
  suspect you chose to convict

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Further Applications of Southern Blotting

• Southern Blotting also enables specific DNA
  comparison and does not leave the Gel
  electrophoresis with too many fragment points
• With genomic libraries, groups and subgroups
  references
• With nucleic acid hybridization Specifies and
  individualizes results

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Mitochondrial DNA

• Mitochondrial DNA is only passed on through the
  mother due to the ability of the egg to supply a
  suitable cytoplasm for the developing human
• The egg during fertilization also cuts off the
  flagella tail cell which contains the paternal
  mitochondria
• Therefore, only maternal mitochondria are passed
  on, and each mitochondria contains separate
  extra-nucleic DNA

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Mitochondrial DNA in Anastasia Romanoff- Anna
Anderson case

• Due to mitochondrial evidence Anna Anderson could
  not have been the Grand Duchess Anastasia
• Dr. Peter Gill stated, If one accepts that this
  sample is from Anna Anderson, then it is almost
  impossible that she could have been Anastasia.
• Through mitochondrial evidence Dr. Gill was able
  to conclude definitively that the two were not
  related, due to their unrelated band lengths and
  matches as seen in previous examples
• He further stated, the sample said to have come
  from Anna Anderson could not be associated with a
  maternal relative of the empress or Prince
  Philip. That is definite.