Theme 4: Applica.ons of DNA Replica.on
12.3 Isola+on, Iden+fica+on, and Sequencing of DNA
Fragments
• Used to determine whether a gene1c risk factor for diabetes has been
inherited, whether blood at a crime scene matches that of a suspect,
whether a variety of rice or wheat carries a gene1c factor for insect
resistance, and how closely two species of organisms are related
12.3.1 The polymerase chain reac+on selec+vely amplifies
regions of DNA
• Polymerase Chain Reac1on (PCR): Developed by Mullis; Allows a
targeted region of a DNA molecule to be replicated (i.e. amplified) into
as many copies as desired; Selec1ve and highly sensi1ve process
◦ Used to amplify and detect small amounts of nucleic acids (like
HIV in blood-bank supplies) and study DNA samples as small as
those leM by a smoker's lips on a cigareOe
• A sample may be as small as a single molecule of DNA
• PCR occurs in a small plas1c tube containing a solu1on of:
◦ Template DNA- At least one molecule of double-stranded DNA
containing the region to be amplified serves as the template for
amplifica1on
◦ DNA Polymerase- Used to replicate the DNA
◦ All Deoxynucleoside Triphosphates- Deoxynucleoside
triphosphates with the bases A, T, G, or C are needed as building
blocks for the synthesis of new DNA strands
◦ Two Primers- Two short sequences of single-stranded DNA are
required for the DNA polymerase to start synthesis
• Enough primer is added so that the number of primer DNA
molecules is much greater than the number of template
DNA molecules
, • Primer sequences are oligonucleo+des produced by
chemical synthesis
• 20-30 nucleo1des long
• Base sequences are complementary to the ends of the
region of template DNA to be amplified
▪ i.e. Primers flank the specific region of DNA
• 3ʹ end of each primer must be oriented toward the region to
be amplified, so that when DNA polymerase extends the
primer, it creates a new DNA strand complementary to the
targeted region
▪ Because the 3ʹ ends of the primers both point toward
the targeted region, one of the primers pairs with one
of the template strands and the other pairs with the
other template strand
◦ Denatura+on- Solu1on is heated to a temperature just shy of
boiling so that the H-bonds between complementary bases break
◦ Annealing- Possible because of the excess of primer molecules
◦ Extension- Solu1on is heated to the op1mal temperature for DNA
polymerase
• PCR usually occurs in a process of 25-35 cycles
◦ In each cycle, the number of copies of the targeted fragment is
doubled (i.e. First round results into 2 copies then, 4, 8, 16, 32, 64,
128, etc.)
• DNA polymerase enzymes from most species irreversibly lose both
structure and func1on at the high temperature needed to separate the
strands of DNA
◦ So, we use DNA polymerase from the bacterial species, Thermus
aqua+cus, that lives at the near–boiling point of water in natural
hot springs
• Known as Taq polymerase
• Can alter the 1me of each cycle, temperatures, number of cycles and
other variables
• The fact that DNA polymerase from a bacterium that lives in hot springs
can be used to amplify DNA from any organism is further evidence for
the conserved func1on and evolu1on early in the history of life of this
enzyme
12.3 Isola+on, Iden+fica+on, and Sequencing of DNA
Fragments
• Used to determine whether a gene1c risk factor for diabetes has been
inherited, whether blood at a crime scene matches that of a suspect,
whether a variety of rice or wheat carries a gene1c factor for insect
resistance, and how closely two species of organisms are related
12.3.1 The polymerase chain reac+on selec+vely amplifies
regions of DNA
• Polymerase Chain Reac1on (PCR): Developed by Mullis; Allows a
targeted region of a DNA molecule to be replicated (i.e. amplified) into
as many copies as desired; Selec1ve and highly sensi1ve process
◦ Used to amplify and detect small amounts of nucleic acids (like
HIV in blood-bank supplies) and study DNA samples as small as
those leM by a smoker's lips on a cigareOe
• A sample may be as small as a single molecule of DNA
• PCR occurs in a small plas1c tube containing a solu1on of:
◦ Template DNA- At least one molecule of double-stranded DNA
containing the region to be amplified serves as the template for
amplifica1on
◦ DNA Polymerase- Used to replicate the DNA
◦ All Deoxynucleoside Triphosphates- Deoxynucleoside
triphosphates with the bases A, T, G, or C are needed as building
blocks for the synthesis of new DNA strands
◦ Two Primers- Two short sequences of single-stranded DNA are
required for the DNA polymerase to start synthesis
• Enough primer is added so that the number of primer DNA
molecules is much greater than the number of template
DNA molecules
, • Primer sequences are oligonucleo+des produced by
chemical synthesis
• 20-30 nucleo1des long
• Base sequences are complementary to the ends of the
region of template DNA to be amplified
▪ i.e. Primers flank the specific region of DNA
• 3ʹ end of each primer must be oriented toward the region to
be amplified, so that when DNA polymerase extends the
primer, it creates a new DNA strand complementary to the
targeted region
▪ Because the 3ʹ ends of the primers both point toward
the targeted region, one of the primers pairs with one
of the template strands and the other pairs with the
other template strand
◦ Denatura+on- Solu1on is heated to a temperature just shy of
boiling so that the H-bonds between complementary bases break
◦ Annealing- Possible because of the excess of primer molecules
◦ Extension- Solu1on is heated to the op1mal temperature for DNA
polymerase
• PCR usually occurs in a process of 25-35 cycles
◦ In each cycle, the number of copies of the targeted fragment is
doubled (i.e. First round results into 2 copies then, 4, 8, 16, 32, 64,
128, etc.)
• DNA polymerase enzymes from most species irreversibly lose both
structure and func1on at the high temperature needed to separate the
strands of DNA
◦ So, we use DNA polymerase from the bacterial species, Thermus
aqua+cus, that lives at the near–boiling point of water in natural
hot springs
• Known as Taq polymerase
• Can alter the 1me of each cycle, temperatures, number of cycles and
other variables
• The fact that DNA polymerase from a bacterium that lives in hot springs
can be used to amplify DNA from any organism is further evidence for
the conserved func1on and evolu1on early in the history of life of this
enzyme
