DNA Fingerprinting

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DNA Fingerprinting

• F.6Sc
• Cherry Kwong, Vincy So,
• Annie Wong, Bobo Lau

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• Principle of DNA fingerprinting

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Something about structure of DNA as we all know

• DNA presents in every cells except red blood
  cells.
• DNA comprises a double helix structure with 2
  strands of polynucleotides.
• Each nucleotide is composed of a pentose, a
  phosphate group and a nitrogenous base.
• There are 4 kinds of nitrogenous bases, namely,
  Adenine (A), Guanine (G). Thymine (T), Cytosine
  (C).

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How DNA determines our characteristics?

• DNA nucleotides sequences determines what amino
  acids are we gong to produce by forming codons.
• A codon is a 3-unit-nucleotides group (eg. TAC,
  TGC) and each represents the amino acid to be
  produced.
• Determining the production in turn determines the
  proteins we possess and once again, in turn,
  determines our characteristics.

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tandem repeats

• In fact, only 10 of our DNA sequence is used for
  coding our essential proteins for life, these 10
  is identical in everyone.
• There remaining 90 consists of sequence of
  nucleotides repeated for many times and is called
  tandem repeats.
• Tandem repeat is a short sequence of about 17 bp
  repeated (between 70- 450 times) in a
  head-to-tail fashion at a specific chromosomal
  locus

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Uniqueness of tandem repeats

• Everyone has their own sequence of tandem repeats
  units and the no. of repeated units varies.
• The chance for 2 unrelated persons having the
  same tandem repeats pattern is 1 in a million
  billion!!
• However, marked similarities are found in related
  individuals.

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DNA fingerprinting based on tandem repeats
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Related individuals

• It is because these highly variable tandem
  repeats are inherited by the offspring with
  complement of genes.
• For instance, S2 (the adopted and non-biological
  son of MOM and DAD) must not inherit any pattern
  from MOM and DAD. While D1, D2, S1 are all
  biological daughters and sons of MOM and DAD
  (thou they have different mothers or different
  fathers), their DNA show similar patterns as MOM
  or DAD do.

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On forensic science

• So, by comparing the DNA tandem repeats of
  individuals obtained from evidence, we can find
  out the offender.
• For instance, if the tandem repeats of suspect1
  matches the DNA tandem repeats on evidences, he
  would have a very high chance to be the offender.

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DNA fingerprinting

• The above technology using tandem repeats of
  individuals to identify individuals is known as
  DNA fingerprinting.

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Procedure of DNA Fingerprinting

• 1. Evidence
• DNA is extracted from blood, hair..etc
• 2. Fragmentation
• cut the DNA into fragments
• 3. Separation
• gel electrophoresis

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Procedure of DNA Fingerprinting

• 4. X-ray
• the separated DNA is transferred to a
• nylon membrane and radioactively
• treated
• 5. Autorad
• X-ray is developed, producing a pattern
• of bands that look like a bar code

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Fragmentation- RFLPs

• Restriction Fragment Length Polymorphism
• organisms may be differentiated by analysis of
  patterns derived from cleavage of their DNA
• if two organisms differ in the distance between
  sites of cleavage of a particular restriction
  endonuclease, the length of the fragments
  produced will differ when the DNA is digested
  with a restriction enzyme.

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Restriction endonucleases

• enzymes that cleave DNA molecules at specific
  nucleotide sequences depending on the particular
  enzyme used.
• If molecules differ in nucleotide sequence,
  fragments of different sizes may be generated.
  The fragments can be separated by gel
  electrophoresis.

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Fragmentation -PCR

• Polymerase Chain Reaction
• to amplify a specific DNA sequence to millions of
  times in just a few hours
• the PCR product can de detected by gel
  electrophoresis
• Increase efficiency in performing DNA
  fingerprinting

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Fragmentation -PCR

• During PCR, high temperature is used to separate
  the DNA molecules into single strands and
  synthetic sequences of single-stranded DNA which
  serve as primers
• Two different primer sequences are used to
  bracket the target region to be amplified.

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Fragmentation -PCR

• 1. at 94-96 ?
• the DNA is denatured into single strands
• 2. at 50-65?
• the primers anneal to their
• complementary sequences on the
• target sequence

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Fragmentation -PCR

• at 72?
• the DNA polymerase binds and extends a
• complementary strand from each primer
• Animation

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Fragmentation -PCR
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Fragmentation -PCR
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Separation Gel electrophoresis

• GelAgarose Gel
• Electrical charge is applied to the gel, with the
  positive charge at the bottom and the negative
  charge at the top
• DNA is slightly negatively charge
• Pieces of DNA will be attracted towards the
  bottom of the gel

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Separation Gel electrophoresis

• Smaller pieces able to move quickly and thus
  further towards the bottom than the larger pieces
• So smaller pieces at the bottom and larger pieces
  at the top

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Separation Gel electrophoresis
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Overall procedure

• http//www.dnalc.org/resources/BiologyAnimationLib
  rary.htm
• NOVA Online Killer's Trail DNA Fingerprint
  Lab

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Application of DNA Fingerprinting

• 1.Criminal Identification Forensics
• DNA fingerprints can be used as biological
  evidence
• Strands of DNA can be found on hair, blood or
  semen.

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• DNA isolated from those evidence can be compared
  through VNTR patterns.
• Useful in solving crimes like murder and rape.
• Example The sex scandal of President Clinton
  with Monica Lewinsky
• Double murders of O.J. Simpson in 1995.

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• 2. Breeding Program
• Breeders traditionally use the phenotype to judge
  the genotype of a plant or an animal
• For example, homozygous dominant genotype AABB is
  desirable.
• Difficult to distinguish homozygous or
  heterozygous dominance from appearance.

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• DNA fingerprinting allows a precise and accurate
  determination of genotype
• Offspring from the selective mating of superior
  animals are like more likely to inherit desirable
  characters like strong cardiopulmonary capacity
  and speed.
• Useful in breeding race horses and hunting dogs

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• 3.Parentage tests
• determine if the alleged father of a child is the
  biological father
• The child (C) will share one band with the
  biological mother (M) and one band with alleged
  father 1 (AF1), the biological father. No bands
  are shared between the child and alleged father
  2 (AF2), the excluded male.

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• 4.Detection of Aids

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• add the blood with the mans DNA, with RNA of
  HIV, together with "reverse transciptase" which
  turns RNA into DNA fragments into the PCR machine
  for amplification
• put them into the gel electrophoresis
• compare the band of HIV "DNA" with the bands form
  by the mans blood

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Advantages of DNA Fingerprinting

• 1.Unsurpassed discriminatory potential
• Complete blood group testing allows discrimiation
  of one person in several thousand and HLA typing
  one in several million
• DNA typing can routinely provide exclusion
  probabilities on the order of one in billions

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• 2.Exquisite sensitivity
• DNA can be amplified
• smaller sample sizes are adequate
• allows rather small samples to be split and
  submitted for testing to more than one laboratory

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• 3.Application to any body tissue
• DNA testing can be conducted with any sample
  having nucleated cells
• For example hairs, semen, urine and saliva

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• 4.DNA is stable in comparison to proteins
• resistant to degradation by common environmental
  insults
• DNA is also long-lived in comparison to protein

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Disadvantages of DNA Fingerprinting

• Problems with determining probability
• DNA fingerprinting is not 100 assured
• VNTR are results of genetic inheritance
• not distributed evenly across all populations
• cannot have a stable probability of occurrence.
• Due to allele frequencies in different population
  or ethnics groups, the probability of match can
  range from 1 in 20 to 1 in 2 billion.

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• Occurrence of certain VNTR pattern depends on an
  individuals genetic background.
• Big problem in determining the VNTR patterns of
  heterogeneous genetic composition of interracial
  individuals
• For example, the frequency of a specific allele
  may be 4 in Asians instead of 1 as it is in
  Northern Europeans.

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• 2. Technical difficulties in DNA fingerprinting
• Errors in the hybridization and probing process
  in carrying out DNA fingerprinting
• Some analysis of DNA sample involves
  amplification of sample.
• A tiny amount of skin cell from the lab
  technician can cause profound error.

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THE END