amplification by lanyuehua

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									Amplification
         Polymerase Chain Reaction
•   PCR – Polymerase Chain Reaction is an enzymatic process in which a
    specific region of DNA is replicated over and over again to yield many
    copies of a particular sequence.

•   PCR was developed in 1985 by Kary B. Mullis, who was awarded the 1993
    Nobel Prize in chemistry for his work.

•   The act of copying DNA in the laboratory

•   Works similar to the way your own cells replicate their DNA

•   With nuclear DNA testing STRs (Short Tandem Repeats) are copied
               What are STRs?
•   Microsatellites called Short Tandem Repeats
•   Highly repetitive sequences
•   2-6 bp repeat unit
•   Polymorphic regions of DNA
    – Number of repeat units differ between chromosomes as
      well as between individuals
      • polymorphisms may result from DNA recombination
        during meiosis
      • polymorphism may also be caused by replication
        slippage
Fluorescent
 dye label                AATG    AATG       AATG




                              7 repeats


                                 8 repeats
      the repeat region is variable between samples while the
      flanking regions where PCR primers bind are constant
 Homozygote = both alleles are the same length
 Heterozygote = alleles differ and can be resolved from one another
   Primer positions define PCR product size
     Why STRs are Preferred
        Genetic Markers
•   Rapid processing is attainable
•   Abundant throughout the genome
•   Highly variable within various populations
•   Small size range allows multiplex development
•   Allelic ladders simplify interpretation
•   PCR allows use of small amounts of DNA material
•   Small product size compatible with degraded DNA
     TPOX                 Positions of STR Markers
         D3S135
                          on Human Chromosomes
           8
                                            TH01
                           D8S1179
                D5S818                             VWA
              FGA        D7S820
   D2S1338      CSF1PO




                                            AME
D13S31                                       L Sex-typing
  7         D16S53       D19S43 D21S11
                     D18S51
         Penta E
               9            3
                                                   AME
                                                    L
                                  Penta D
               PCR Amplification
• Components of PCR amplification Master Mix
   – Template DNA (1-10ng)
   –   Primers
   –   Deoxynucleotide triphosphates dNTPs
   –   MgCl2
   –   DNA polymerase
   –   Buffer BSA
                    Template DNA
• The PCR sample may be single- or double-stranded DNA or
  RNA.

• 0.5 -2ng of DNA is targeted

• Approximately 20,000 target copies are optimal.

• Preferential amplification can occur if target amount is too low.
               PCR Amplification
• Components of PCR amplification Master Mix
   – Template DNA (1-10ng)
   – Primers (0.1-1.0 0mM)
   –   Deoxynucleotide triphosphates dNTPs (200mM each)
   –   MgCl2 (1.2-2.5mM)
   –   DNA polymerase (0.5-5.00mM)
   –   Buffer (Tris 10-50mM pH8.3, KCl 50mM)
   –   BSA (100mg/mL)
                             Primers
• PCR primers are oligonucleotides hybridizing to opposite strands
  and flanking the region of interest in the target DNA.
• Several variables must be taken into account when designing
  PCR Primers.
• Primer Design Considerations:
   –   Primer length
   –   Melting Temperature (Tm)
   –   Annealing temperature
   –   Complementarity
   –   Primer structure
   –   Sequence content
                         Primer Design
• Primer length
   – typically 18-30 bases long to increase specificity and decrease cross-
     hybridization


• Melting Temperature (Tm)
   – Measure the stability of the primer-template DNA. Temperatures in the range of
     52-58°C produce better results. The Tm is affected by the primer length and GC
     content.
   – Tm differnece between primers should be less than 5°C in order to avoid
     preferntial amplification


• Annealing Temperature (Ta)
   – Typically 5°C below the lowest Tm of the primers. Optimal Ta yields less non-
     specific amplification
                       Primer Design
• Complementarity
  – Self- complimentarity primers should be avoided.(no intra-primer homology
    beyond 4 base pairs) to prevent the formation of primer dimers called self-
    dimer.
  – Primers with complimentarity with other primers (especially at the 3’end)
    need to be avoided to prevent the formation of primer dimers called cross-
    dimers .
                       Primer Design
• Primer structure and sequence content
   – G/C Content
      • A 40%-60% G/C content is recommended for both primers.
      • Greater than 3 repeats of G or C at the 3’ end of the primer (GC clamps)
        should be avoided to prevent the formation of primer-dimers .
   – Primers with Secondary structure should be avoided

   – Primers with long runs of the same base should be avoided
      • this prevent “breathing” of the primer, and therefore causes mispriming
  Other Considerations for Primers
• Degenerate primers

• Fluorescently labeled primers
            Degenerate Primers

• Point mutations at the primer binding locations may
  cause null alleles
• Degenerate primers are a set of primers that have
  several options in the sequence allowing for point
  mutations
• When you type a sample with one kit it will appear as a
  homozygote if a null allele is present, but when typed
  with degenerate primers the null allele may be revealed
  showing that it was truly a heterozygote.
 Methods for Fluorescently Labeling DNA
• Intercalating Dyes (post-PCR)
• Dye-labeled nucleotide insertion during PCR
• Dye-labeled primer insertion during PCR
Fluorescent Labeling of DNA with Primers

• Dyes are attached to one primer in a pair used to amplify a STR
  marker

• Dyes are coupled to oligonucleotides (primers) through NHS-esters
  and amine linkages on the 5’end of the primer:
               » Dye-(CH2)6-primer

• Dye-labeled oligonucleotides are incorporated during multiplex PCR
  amplification giving a specific color “tag” to each PCR product

• PCR products are distinguished using CCD imaging on the 310
              PCR Amplification
• Components of PCR amplification Master Mix
   – Template DNA
   – Primers
   – Deoxynucleotide triphosphates dNTPs
     (20 -200mM each)
   –   MgCl2
   –   DNA polymerase
   –   Buffer
   –   BSA
    Deoxynucleotide Triphosphates
• It is important to keep the four dNTPs (dATP, dTTP, dCTP and
  dGTP) in equal concentrations (20 µM-200 µM) to minimize mis-
  incorporated nucleotides.

• Lowering the dNTP concentration can improve fidelity.
             PCR Amplification
• Components of PCR amplification Master Mix
   – Template DNA
   – Primers
   – Deoxynucleotide triphosphates dNTPs
   – MgCl2 (0.5 -2.5mM)
   – DNA polymerase
   – Buffer
   – BSA
                          MgCl2
• MgCl2 concentration may affect the following:
   – Primer annealing
   – Strand dissociation temperature of template
   – Strand dissociation temperature of PCR product
   – Product specificity
   – Formation of primer-dimers
   – Enzyme activity and fidelity
                             MgCl2
• Generally, increasing the free magnesium concentration increases
  yield and decreases specificity and fidelity.



• When using AmpliTaq® DNA Polymerase, too little free magnesium
  will result in little or no PCR product, and too much free magnesium
  ion may produce a variety of unwanted products and promote
  misincorporation. The optimal amount is in the range of 0.5 to 2.5mM.
               PCR Amplification
• Components of PCR amplification Master Mix
   –   Template DNA
   –   Primers
   –   Deoxynucleotide triphosphates dNTPs
   –   MgCl2
   – DNA polymerase (0.5-5.00mM)
   – Buffer
   – BSA
    AmpliTaq ® Gold - DNA Polymerase

• Originally isolated from the thermophilic eubacterium, Thermus
  aquaticus.
• It is now supplied as a recombinant enzyme from E. coli.
• The enzyme is highly purified and is free of nonspecific endo- or
  exonucleases.
• It is a highly processive 5'–3' DNA polymerase which lacks 3'– 5'
  exonuclease activity.
• It is chemically modified to be inactive until the reactions reaches
  95 oC for 10 or 11 minutes (Hot start PCR) which improves the
  specificity, sensitivity, and yield of PCR.
• Allows for room temperature amplification set up.
                        Hot Start PCR
•   Why Hot start PCR
    – Regular DNA polymerases exhibit some activity below their
      optimal temperature. Therefore during set up primers can
      anneal non-specifically to the template DNA at room
      temperature and non-specific products may result. Also at low
      temperatures primers may bind to other primers forming primer
      dimers which can be preferentially amplified over template
      DNA.

    With Hot start PCR the DNA polymerase does not show any activity
       at room temperature. So there is no extension of non specific
       products or primer dimers.
               PCR Amplification
• Components of PCR amplification Master Mix
   –   Template DNA
   –   Primers
   –   Deoxynucleotide triphosphates dNTPs
   –   MgCl2
   –   DNA polymerase
   – Buffer (pH8.3)
   – BSA
          GeneAmp® PCR Buffers
• The GeneAmp 10X PCR Buffer is composed of 500 mM potassium
  chloride, 100 mM Tris-HCl (pH 8.3 at room temperature), 15 mM
  magnesium chloride and 0.01% (w/v) gelatin (BSA).


• The recommended dilution buffer for AmpliTaq® Gold DNA
  Polymerase has pH 8.3 at room temperature which will decrease to
  pH 6.9 when heated to 95oC.
               PCR Amplification
• Components of PCR amplification Master Mix
   –   Template DNA
   –   Primers
   –   Deoxynucleotide triphosphates dNTPs
   –   MgCl2
   –   DNA polymerase
   –   Buffer
   – BSA (100mg/mL)
            BSA and other Additatives
In some cases, adding the following compounds can enhance the
efficiency or specificity of PCR:
       Betaine (0.5–2 M)
       Bovine serum albumin (BSA; 100 ng)
       Detergents
       Dimethylsulfoxide (DMSO; 2–10%) (v/v)
       Gelatine
       Glycerol (1–5%) (v/v)
       Pyrophosphatase (0.001–0.1 units/reaction)
       Spermidine
                PCR Instrumentation

• The instrument used to perform PCR is the thermal
  cycler.

• Thermal cyclers allow for the rapid and accurate heating
  and cooling of DNA samples which is crucial to PCR.

• Modern thermal cyclers have a heated lid this prevents
  PCR reagents from condensing on the top of the tube
  during the temperature cycling.
                     PCR Cycling Process
Step                    AmpFlSTR Kits     PowerPlex Kits

Initial incubation      95°C for 11 min   95°C for 11 min, 96°C for 1min
Cycling                 28 Cycles         32 Cycles

    Denaturation        94°C for 1 min    94°C for 30 secs (C1-10)
                                          90°C for 30 secs (C11-32)

    Anneal              59°C for 1 min    60°C for 30 secs

    Extend              72°C for 1 min    70°C for 45 secs

Final extension         60°C for 45 min   60°C for 30 min

Final hold              4°C               4°C
              Polymerase Chain Reaction (PCR)
                          Process
    5’                3’


    3’                5’

                  Denaturation (94oC)
                                 5’                   3’
    3’                5’                                       25 – 35
                               Annealing (59oC)                cycles
         5’ 3’
                                 5’                       3’
    3’                5’
                                                  3’ 5’
                                Extension   (72oC)
         5’
                                 5’                       3’
1
    3’                5’
                                                     5’
                  PCR Process
• Initial incubation
   – Performed at approximately 95°C for 11 min


   – Activates AmpliTaq ® Gold
                       PCR Process
• Denaturation (Performed approximately 94°C):
   – During the denaturation, the double strand melts open to single stranded
     DNA, all enzymatic reactions stop (for example: the extension from a
     previous cycle). Incomplete denaturation allows for snapback to occur,
     which reduces product yield.
                       PCR Process
• Annealing (Performed at approximately 59°C) :
   – Ionic bonds are formed between the primers and the template DNA, the
     polymerase can attach and starts copying the template. The higher the
     temperature, the more specific binding occurs
                        PCR Process
• Extension (Performed at approximately 72°C) :
   – This is the ideal working temperature for the polymerase.Taq DNA
     Polymerase can add approximately 35 to 100 base pairs per second.
   – The bases (complementary to the template) are coupled to the primer on
     the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template
     from 3' to 5' side, bases are added complementary to the template)
                  PCR Process
• Final Extension:
   – Performed at approximately 60°C for 45 min
   – Ensures complete adenylation
   – If there is excessive –A , an additional extension cycle
     can be performed if allowed by the laboratory SOPs.
      Polymerase Chain Reaction

                              One copy of DNA
                  One cycle


                                  Two copies of DNA

          Second cycle


                                      Four copies of DNA


Third cycle


                                         Eight copies of DNA
               Cycles
Cycle Number    Copy Number
     1                     2
     2                     4
     3                     8
     4                     16
     5                     32
     N                     2n
          Controls Used in PCR
• Two main controls used in PCR are a Positive and Negative
  control

• Positive Control – DNA supplied by makers of the
  amplification kits whose concentration is known. This is used
  to ensure there are no problems with the PCR process
  whether it be from the reaction components or the
  instrument itself.
• Negative control – Contains all the PCR reagents but no
  DNA. This is used to ensure that there is no contamination in
  the reagents or that none was introduced during
  amplification set up.
        Critical Factors to remember for
             successful amplification
•   Wear gloves.
•   Change gloves to prevent contamination.
•   Use aseptic technique.
•   Use only plugged tips.
•   Use only sterile DI water.
•   Taq is kept in the DNA Free freezer. When not in use it shall be
    kept in the freezer. Place back in freezer as soon as possible.
•   Protect the Primer from light while in use. The fluorescent dyes
    attached to the primers are light sensitive.
•   Master MIX should be prepared just before use
•   Dilutions and calculations for proper template amount should be
    performed prior to amplification set up.
        Critical Factors to remember for
             successful amplification
• Tubes should be properly labeled.
• Only one tube opened at a time
• Work in a hood if possible, if not use a face mask and avoid
  talking, coughing or sneezing over tubes.
• Any human DNA introduced into the tube will be amplified so if
  your DNA or DNA from someone else gets into tube it will be
  amplified.
• Develop a method to keep track of where you are during the set
  up process so if a distraction occurs you can easily find where
  you stopped. This avoids missing tubes or double loading or
  putting wrong sample in wrong tube.

								
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