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					      DNA Profiling using Short
         Tandem Repeats

          This slide show includes information from the following website:

WWW.CRIMESCENE.COM thanks John M. Butler, Ph.D at the NIST Biotechnology
 Division for his help and permission to include information and graphics in this
            DNA Profiling using STRs:
                 An Overview
    STR analysis of DNA samples is a DNA profiling technique that uses PCR (polymerase
    chain reaction) to copy samples of DNA at distinct locations and analyze their sizes (the
    size of the DNA piece being the basis of comparison between samples).
•   STRs are Short Tandem Repeats of patterns of nucleotides spread throughout our DNA

                     AATG AATG AATG AATG AATG AATG AATG                         DNA molecule
              7 short, tandem (back to back) repeats of the nucleotide sequence AATG

•   The number of repeats at a certain distinct region (locus, plural=loci) of DNA is highly
    variable from person to person allowing their use in human identity testing
•   The number of nucleotides involved in the repeats can vary between 9 and 80 (called
    variable number of repeats, VNTRs, or minisatellites) or between 2 and 5 (called
    microsatellites, SHORT tandem repeats, STRs)
•   Several loci along our DNA have been identified as possessing STRs (thanks in part to
    the Human Genome Project), and the DNA profiling community has selected 13 regions
    for identity analysis
•   These 13 loci ALL contain 4 nucleotide (tetrameric) repeats
•   Through population studies, the numbers and types (nucleotides involved) of these
    repeats at these loci have been analyzed affording probability estimates in certain
13 STR Loci for DNA Profiling
•13 STR loci have been officially chosen to be used in the Combined
DNA Index System (CODIS) and are scattered among our 23 chromosome
•CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820,
D8S1179, D13S317, D16S539, D18S51, and D21S11
•While not an STR, the AMEL (amelogenin) locus on chromosome 23, the
sex chromosome, is included for gender determination


                                           D8S1179       THO1
                             D5S818                             VWA


       D13S317       D16S539      D18S51        D21S11          AMEL
             DNA Profiling using STR:
                  An Overview
•   Because we have 23 pairs of chromosomes (23 chromosomes from our mother, 23 from
    our father), the loci are actually duplicated; each of the 13 CODIS loci exist on one
    chromosome from our mother and one from our father.
•   Therefore, when analyzing the number of repeats at a certain locus, generally we get 1
    (the number of repeats is the same) or 2 (the number of repeats is different) answers,
    representing the pair. These are known as alleles. We get one allele from each parent.
•   If the number of repeats is the same, we are “homozygous” at that locus. If the number
    of repeats is different, we are “heterozygous” at that locus. Any variety of homozygosity
    or heterozygosity can be present at the 13 loci.
•   Therefore, you can see how STRs can be used for maternity and paternity testing.

                     AATG AATG AATG AATG AATG AATG AATG                  DNA molecule
      7 short, tandem repeats of the nucleotide sequence AATG            from mother

                                                         AATG            DNA molecule
      8 short, tandem repeats of the nucleotide sequence AATG             from father
           Advantages of STR Analysis
•   The allelic variation (number of repeats) of STRs is more easily discernable than other
    techniques (a difference in repeat of just one, or 4 nucleotides, can be seen with current
•   The number of repeats at the STR loci is discrete, meaning from current studies, there
    are a set amount of answers, facilitating interlaboratory comparisons.
     –   E.g., at locus THO1, which is found on chromosome 11, studies have shown that the tetramer
         AATG repeats anywhere from 3 to 14 times. If one strand of DNA (from your mother) contains
         3 repeats and the other (from your father) contains 5 repeats, the profile is dubbed “3,5”. This
         profile is then used for comparison to other DNA samples.
•   Because PCR (polymerase chain reaction) is used to amplify the STR loci, very small
    quantities of DNA are needed (a blood droplet the size of the head of a pin)
•   Because the size of the STR loci are relatively small, the odds that the STR locus will be
    completely intact and therefore available for analysis in a degraded DNA sample (DNA
    that has already been somewhat cut from just being deposited in environments where
    decomposition could occur) are higher
•   Technology allows complete analysis in a matter of hours
Sources of Biological Evidence
•   Blood
•   Semen
•   Saliva
•   Urine
•   Hair
•   Teeth
•   Bone
•   Tissue
                       DNA in the Cell

cell nucleus

     Double stranded
     DNA molecule          Target Region for PCR
                                                      T       A
                                                  C               T
 Orange = A                 T
                                G             C                       C
  Green = T                                           A                   T
                                        G A               T
 Purple = C                 A                         G
 Yellow = G                         AC        IndividualA
                                          T G
                                              nucleotides G A
                                                  (A, T, C, G)
            Steps in DNA Sample Processing
 Sample Obtained from
Crime Scene or Paternity
     Investigation                Biology
            DNA                  DNA                   PCR Amplification
          Extraction          Quantitation          of Multiple STR markers

       Separation and Detection of                Sample Genotype
              PCR Products                         Determination
              (STR Alleles)

     Comparison of Sample                                   Generation of Case
      Genotype to Other                                   Report with Probability
        Sample Results                                       of Random Match

                           If match occurs, comparison
                           of DNA profile to population
                                        DNA 101
•It has been mentioned that the repeats of STRs are composed of nucleotides.
Amazingly, the genetic code (the DNA represented by all our 23 pairs of
chromosomes) is composed of only four nucleotides in a string: Adenine (A),
Thymine (T), Cytosine (C) and Guanine (G). These are the sole letters of the genetic
•Nucleotides are also known as nitrogenous bases, or just “bases”.
•Adenine and guanine are known as the purine nitrogenous bases, while cytosine and
thymine are called the pyrimidine bases; adenine binds only to thymine and cytosine binds
only to guanine.
•In a DNA molecule (on just one chromosome), the structure looks like a twisted ladder, with
the rungs representing the pairs of the nitrogenous bases. Nucelotides are therefore also
termed base pairs, or bps, when talking about the “double stranded” DNA molecule.
•The pattern of these letters constructs genes, that in turn act as templates for proteins, that
in turn help to construct and operate the human body. Yet there is enough variation to make
us all unique. Mind boggling.
 nucleotides                                              C T     A T                     G T
                                                      C                               T
 Orange = A                         TG                                    C
                                            T                                     C               DNA Molecule on one
                                                G A                           T                      chromosome
  Green = T                                                   A   T                           A
                                        C                 G
 Purple = C                                 AC    TG
                                                                      A                   C
                                                                                  G A
 Yellow = G
      The Polymerase Chain Reaction
•The amount of DNA represented in a pure sample dwarfs the amount represented
in just the 13 CODIS loci. Therefore, these regions, and only these regions, need to
be magnified for analysis, and the polymerase chain reaction (PCR) is used as a
molecular Xerox machine just for this purpose.
•PCR employs the use of primers, which are short pieces of single stranded DNA
complementary to areas along a certain piece of DNA that you want to magnify (i.e.
the THO1 locus).
•Using the rule from biology 101 that A binds to T and C to G, primers are designed
(and subsequently synthesized in a laboratory very easily and quickly) using this
rule to bind to a region of DNA just BEFORE the STR region on one DNA strand and
just AFTER the STR region on the complementary DNA strand of the double
stranded DNA molecule on each chromosome (e. g. chromosome 11 for the THO1
locus). for primer binding

    A G C A T A A T T C A A T G A A T G A A T G C G T A C C T A
    T C G T T T T A A G T T A C T T T G T T A C G C A T G C A T

               STR Region in red (3-AATG repeats)   Region for primer binding

                                                                            - - - - Rest of DNA
                                                                            on chromosome 11
                                                                            from one parent
                       DNA Amplification with the
                    Polymerase Chain Reaction (PCR)
•The DNA sample is heated to allow the double stranded DNA to “denature” or become
single stranded, so that the single stranded primers can get in. The reaction is cooled so
that the primers can bind (anneal) to their complementary regions of the sample.
•An enzyme (DNA polymerase, which polymerizes the nucleotides to the primers), along
with spare nucleotides, is added to this mixture, and the enzyme begins to extend the
primers along the regions of interest (i.e. THO1) in complementary fashion, basically
copying the THO1 region and its various number of tetrameric repeats.
•This reaction (denaturing, annealing, extension) is allowed to repeat itself (in a
thermocycler) many times, magnifying the DNA locus, and ONLY that locus, many times.

  Single stranded
 (denatured) DNA
                      A G C A T A A T T C A A T G A A T G A A T G C G T A C C T A
                                                                    C G C A T G C A T
                                        DNA polymerase      A
                                                       G  T
                         Forward primer                               Reverse primer
                                                         T   G   C
                                                    T Spare nucleotides
                      A G C A T A A T T C            CA    A     G C
                       T C G T T T T A A G T T A C T T T G T T A C G C A T G C A T
  Single stranded
 (denatured) DNA
                         DNA Amplification with the
                      Polymerase Chain Reaction (PCR)
  •Because the newly synthesized fragments of DNA are used for the
  second round of synthesis and have one finite end representing the
  beginning of the primer, subsequent cycles will produce an excess of
  ONLY the region of interest (beginning with the start of the forward
  primer to the end of the reverse primer)

  •With 32 cycles, over 1 billion (232) molecules of DNA representing a
  specific locus (and only that locus) are synthesized and now dwarf the
  rest of the DNA of the sample.
   Single stranded
  (denatured) DNA,
  original template      A G C A T A A T T C A A T G A A T G A A T G C G T A C C T A
                         T C G T T T T A A G T T A C T T T G T T A C G C A T G C A T
Newly synthesized DNA

Newly synthesized DNA
                        A G C A T A A T T C A A T G A A T G A A T G C G T A C C T A
                         T C G T T T T A A G T T A C T T T G T T A C G C A T G C A T
   Single stranded
  (denatured) DNA,
  original template
Multiplex STR Analysis

               •   Because the primers of each locus have been
                   stringently designed to be specific for only
                   regions before and after their locus, over 10
                   loci can be copied at once in one tube
               •   Sensitivities to levels less than 1 billionth of a
                   gram of DNA are possible
               •   Different fluorescent dyes are used to
                   distinguish STR loci with overlapping size
               •   Generally, the result (if using 13 STR loci and
                   the sample is pure) is a mixture of as few as 13
                   and as many as 26 PCR products representing
   Original DNA    13 STR loci (13 products if at EVERY locus, the
    Template       individual is homozygous, or at each locus, the
                   same number of repeats (same size) is present
                   – OR – 26 PCR products if at every locus the
                   individual is heterozygous, or at each locus, a
                   different number of repeats (different size) is
PCR Products
               •   If the sample contains a mixture, more pieces
                   will be seen.
  Available Kits for STR Analysis
• Kits make it easy for labs to just add DNA
  samples to a pre-made mix
• 13 CODIS core loci
   – Profiler Plus and COfiler (PE Applied Biosystems)
   – PowerPlex 1.1 and 2.1 (Promega Corporation)
• Increased power of discrimination
   – CTT (1994): 1 in 410
   – SGM Plus™ (1999): 1 in 3 trillion
   – PowerPlex ™ 16 (2000): 1 in 2 x 1017
                           STR Analysis
•Once PCR has successfully been completed using any of the kits available, the products
must be analyzed
•One method used is capillary electrophoresis (CE), which involves injecting the PCR
products through a thin capillary
•Smaller sized fragments will move faster, and thus reach the fluorescence detector first.
•The wavelengths emitted by each fluorescent dye is different and can be monitored.
•Because it is known which fluorescent dyes are used for each locus, and it has been
controlled that loci containing similar size fragments use DIFFERENT dyes, each product
can be identified as it is detected.
•Standards are included that contain the known sizes produced at the various loci
• The fluorescence detection results in peaks representing different sizes and intensities
•The amelogenin locus, while not an STR, is included for gender determination
     •A female is homozygous at the AMEL locus (X, X) and thus will display one peak
     •A male is heterozygous at the AMEL locus (X, Y) and thus will display two peaks
                 An Example Forensic STR Multiplex Kit

                                 AmpFlSTR® Profiler Plus™
                                       Kit available from PE Biosystems (Foster City, CA)

                        100 bp                    200 bp                 300 bp               400 bp
                                                   Size Separation
     Color Separation

                                 D3         vWA             FGA                   5-FAM (blue) dye

                            A         D8           D21             D18            JOE (green) dye

                                  D5              D13               D7            NED (yellow) dye

                                                                                                    ROX (red)
                                           GS500-internal lane standard
9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction
                            Human Identity Testing with Multiplex STRs
                                                                            AmpFlSTR® SGM Plus™ kit

                                                    Homozygous at THO1 DNA Size (base pairs)
                               fragmentsD3                                Heterozygous at D16
                            amelogenin     D8 TH01 (1 peak)                               Larger
Two different individuals

                                                                          (2 peaks)
                                    D19           VWA D21             D16               fragments
                                                                                 D18       D2

                             amelogenin D3               Male (2 peaks)

                                     D19                 Female (1 peak)
                                             D8      VWA                   D16
                                                                  D21                  D18    D2

                                 Simultaneous Analysis of 10 STRs and Gender ID
                     Example of STR Allele
                                                TH01 Marker

            25                                   Caucasians (N=427)
            20                                   Blacks (N=414)
            15                                   Hispanics (N=414)
             5                                *Proc. Int. Sym. Hum. ID
                                              (Promega) 1997, p. 34
                 6    7   8    9   9.3   10
                     Number of repeats
                     Probability Estimates
•   Referring to the previous slide, the graph represents the frequency of a set of repeats at
    the THO1 locus.
•   While it is known that the number of repeats (comprised of the tetrameric sequence
    AATG) varies from 3 to 14, only the repeats of 6 to 10 are represented here.
•   Generally, if only using this graph as the basis for probability estimates, the frequency of
    each allele (repeat number) compared to the total number of samples used (427 for
    Caucasians, 414 for African Americans and Hispanics) would be used to calculate the
    probability estimate of THAT allele for THAT locus in THAT specific population
•   This is repeated for all other alleles in each population, thus constructing probability
    estimates of specific alleles for each of the 13 CODIS loci for each ethnicity
•   **NOTE** How is a repeat of 9.3 possible???
     –   It has been observed that in some loci, repeats cannot be entirely complete; in a stretch of DNA
         containing an STR, most of the repeats are tetramers (4 nucleotides), but within these, a portion
         of a repeat (e.g. 2 or 3 of the 4 nucleotides of the repeat) may be present
     –   Because the 3-nucleotide fragment occurs within the stretch of 4-nucleotide repeats, it is
         included as part of the STR, but the notation is different
     –   In the above example, the number of repeats is represented as 9.3, because there are 9 intact
         tetrameric repeats with one partial repeat of 3 nucleotides “.3”.
                Probability Estimates

Databases of the frequencies of each number of repeats of each locus in a
given population are used to calculate probabilities. Probability estimates
at each locus are multiplied by estimates at other loci to afford an overall
probability estimate

Because as many as 13 loci are analyzed and compared to questioned
samples, probability estimates can reach over 1 in a trillion, eliminating
basically everyone on the globe, underscoring the power of STR analysis.

Therefore, the more loci examined, the more powerful the analysis.

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