PCR (Polymerase Chain Reaction)

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Making DNA Molecules
Chapter 13
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Learning Outcomes

• Describe the process of semiconservative DNA
  replication in cells and compare and contrast
  this method with DNA synthesis in the laboratory
• Discuss the uses of synthesized oligonucleotides
  and identify the attributes of good primers
• Explain the steps of PCT and discuss the
  components and optimization of the process
• Discuss the function of a thermal cycler and how
  PCR results are visualized
• Describe applications of PCR technology,
  including uses in the field of forensics

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13.1 Making DNA Molecules DNA Synthesis

• A DNA molecule, at any given moment, could be
  involved in
• DNA replication
• Transcription

DNA and Chromosomes
DNA molecules directly code for all the RNA and
protein molecules that a cell synthesizes. The 44
chromosomes in human cells are actually 22
homologous pairs, plus 2 sex chromosomes.
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DNA Replication
A human body is estimated to have over 20
trillion cells. These cells all originate from a
single fertilized egg cell by means of DNA
replication.
DNA Template
A template DNA is the strand from which a new
strand is synthesized.
Primer
A primer is a short piece of DNA or RNA that is
complementary to a section of template strand.
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Nucleotides
Nucleotide triphosphates are the reactants used
as the sources of A, C, G, and T for the new
strand.
DNA Polymerase
Polymerase builds large molecules (polymers) from
smaller molecules (monomers).
Reaction Buffer
Reaction buffer is used to maintain the pH of the
synthesis reaction.
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Vocabulary

• Homologous pairs two matching chromosomes
  that have the same genes in the same order
• DNA replication process by which DNA molecules
  are duplicated
• in vivo referring to an experiment conducted in
  a living organism or cell literally in living
• Helicase enzyme that functions to unwind and
  unzip complementary DNA strands during in vivo
  DNA replication
• Topoisomerase enzyme that acts to relieve
  tension in DNA strands as they unwind during in
  vivo DNA replication
• RNA primase enzyme that adds primers to
  template strands during in vivo DNA replication
• Primer a short piece of DNA or RNA (15 35
  bases) that is complementary to a section of
  template strand and acts as an attachment and
  starting point for the synthesis strand during
  DNA replication
• DNA polymerase enzyme that, during DNA
  replication, creates a new strand of DNA
  nucleotides complementary to a template strand
• RHase H enzyme that functions to degrade RNA
  primers, during in vivo replication, that are
  bound to DNA template strands

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Vocabulary

• in vitro synthesis any synthesis that is done
  wholly or partially outside of a living organism
  (eg, PCR) literally, in glass
• Probes fluorescently labeled DNA or RNA
  sequences (oligonucleotides) that are used for
  gene identification
• DTT abbreviation for dithiothreitol, a reducing
  agent that helps stabilize the DNA polymerase in
  DNA synthesis, PCR, and DNA sequencing reactions
• Template strand of DNA from which a new
  complementary strand is synthesized
• dNTP abbreviation for nucleotide triphosphates,
  which are the reactants used as the sources of A,
  C, G, and Ts for a new strand of DNA
• dATP abbreviation for deoxyadenosine
  triphosphate, the cells source of adenine (A)
  for DNA molecules
• dCTP abbreviation for deoxycytidine
  triphosphate, the cells source of cytosine (C)
  for DNA molecules
• dGTP abbreviation for deoxyguanosine
  triphosphate, the cells source of guanine (G)
  for DNA molecules
• dTTP abbreviation for deoxythymidine
  triphosphate, the cells source of thymine (T)
  for DNA molecules
• Reaction buffer buffer in PCR that is used to
  maintain the pH of the synthesis reaction

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13.1 Review Questions

• How many chromosomes does an E. coli cell
  contain? How many chromosomes does a human body
  cell contain?
• What are homologous pairs, and where do they come
  from?
• Name six enzymes involved in in vivo DNA
  replication.
• How is in vitro DNA synthesis in a test tube
  different than in vitro DNA synthesis in an
  automated synthesis?

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13.2 DNA Synthesis Products

• DNA is commonly synthesized for these
  applications
• Probes
• Primers
• PCR amplification

Probes
Probes are relatively short pieces of DNA (or
RNA) with a nucleotide sequence complementary to
another sequence being searched for.
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Blotting
Samples are transferred from the gel to a
membrane or specially treated paper.
Microarrays
Microarrays are assemblies of large numbers of
samples of DNA, or even RNA samples.
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Constructing Primers
Primers are constructed to recognized a
particular section of DNA. This is called primer
design.
PCR Amplification
Primers are used when trying to mark, identify,
or amplify a piece of DNA.
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Vocabulary

• Amplification increase in the number of copies
  of a particular segment of DNA, usually as a
  result of PCR
• Cross-linker instrument that uses UV light to
  irreversibly bind DNA or RNA to membrane or paper
• Microarry scanner instrument that assesses the
  amount of fluorescence in a feature of a
  microarray
• Primer design process by which a primer
  sequence is proposed and constructed

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13.2 Review Questions

• What is it called when DNA samples are
  transferred to a membrane for staining or
  probing?
• How are probes used in microarrays?
• Design a primer that would be good for
  recognizing the beginning of the following
  sequence of interest. Describe why your primer
  is a good one.
• 3ACACAGGATACGTGCTGCTCAATGCCATGATAGCCGGTCACAAGC-
• TAATCCGATTTCGCGCAAATTCCTAAATTCGCTAAAGC-
• GAATCTTCAGGAAGGAACCCCGAAGGCCTTTT-5, and so on.

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13.3 Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a method by
which millions of copies of a DNA segment can be
synthesized in a test tube in just a few hours.
Performing a PCR Reaction

• Reaction buffer Maintains pH
• Forward primers Recognize one end of the
  fragment to be amplified
• Reverse primers Recognize the other end of the
  fragment to be amplified
• Taq polymerase Special DNA polymerase that
  remains active at very high temperatures
• dNTPs The four deoxynucleotides (A, C, G, T)
• Magnesium chloride (MgCl2) Necessary cofactor
  for polymerase activity

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Cyling Program
The cycling program chosen depends on the type of
sample to be amplified.
Challenges in PCR Technology

• DNA samples are often compromised.
• Concentration of reagent, and the time and
  temperatures of the thermal cycling program may
  affect the results.

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Vocabulary

• Primer annealing phase in PCR during which a
  primer binds to a template strand
• Extension phase in PCR during which a
  complementary DNA strand is synthesized
• Optimization process of analyzing all the
  variables to find the ideal conditions for a
  reaction or process

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13.3 Review Questions

• Why is Taq polymerase used in PCR instead of some
  other DNA polymerase?
• What are the three parts to a thermal cycling
  reaction, and what is the difference in
  temperature between them?
• What is it called when a PCR technician
  determines the best conditions for running a PCR
  protocol?

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13.4 Applications of PCR Technology

• Forensics/criminology
• Missing children/soldiers
• Paternity/maternity cases
• Medical diagnostics
• Therapeutic drug design
• Phylogeny/evolutionary studies
• Animal poaching/endangered species

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DNA Fingerprinting
PCR technology came into public spotlight during
the 1992 O.J. Simpson murder trial.
Forensics
Forensics is the application of biology,
chemistry, physics, mathematics, and sociology to
solve legal problems.
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Vocabulary

• Karyotyping process of comparing an
  individuals karyotype with a normal, standard
  one to check for abnormalities
• VNTRs abbreviation for variable number of
  tandem repeats, sections of repeated DNA
  sequences found at specific locations on certain
  chromosomes the number of repeats in a
  particular VNTR can vary from person to person
  used for DNA fingerprinting
• Forensics application of biology, chemistry,
  physics, mathematics, and sociology to solve
  legal problems including crime scene analysis,
  child support cases, and paternity

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13.4 Review Questions

• Restriction fragment length polymorphism
  technology was formerly used for DNA
  fingerprinting. What technology is currently used
  for DNA fingerprinting?
• For a DNA fingerprint, many PCR targets are used.
  Each target is its own VNTR. What is a VNTR?
• Why would looking for the persons responsible for
  sneaking endangered species (rare birds, for
  example) into the United States be considered a
  job for a forensic scientist?

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Questions and Comments?
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PCR(Polymerase Chain Reaction)
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PCR Polymerase Chain Reaction has many
applications

• PCR is commonly used to produce many copies of a
  selected gene segment or locus of DNA.
• In criminal forensics, PCR is used to amplify
  DNA evidence from small samples that may have
  been left at a crime scene.
• PCR can be used to amplify DNA for genetic
  disease screening

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The PCR ReactionHow does it work?
Heat (94oC) to denature DNA strands Cool (56oC)
to anneal primers to template Warm (72oC) to
activate Taq polymerase, which extends primers
and replicates DNA Repeat 40 cycles
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PCR

• 94 C Denature DNA
• 56 C Anneal Primers to Template
• 72 C Activates Taq Polymerase
• Repeats 31 times

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The PCR ReactionWhat do you need?
What is needed for PCR?

• Template - the DNA to be amplified
• Primers - 2 short specific pieces of DNA whose
  sequence flanks the target sequence
• Forward
• Reverse
• Nucleotides - dATP, dCTP, dGTP, dTTP
• Magnesium chloride - enzyme cofactor
• Buffer - maintains pH contains salt
• Taq DNA polymerase thermophillic enzyme from
  hot springs (Thermus aquaticus)

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What do we use?

• Reagents and supplies Equipment
• and supplies
• Genomic DNA sample (5 µL) P-20 pipette and tips
• Master mix I (10 µL/reaction) Thermal cycler
• 2.5 µL 10x PCR buffer w/o MgCl2
• 0.5 µL dNTPs (10 mM)
• 2.5 µL Forward primer (4pM/ µL)
• 2.5 µL Reverse primer (4pM/ µL)
• 0.15 µL Taq polymerase
• 1.85 µL ddH2O
• Master mix II (10 µL/reaction)
• 0.75 µL MgCl2 (50 mM)
• 9.25 µL ddH2O

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Expected Results of PCR--Example

• Homozygous Alu
• Homozygous Alu
• Heterozygous
• Marker

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Expected Results