Part 2: Lesson 10, 11, 12, 13 Flashcards
(30 cards)
3 categories of Amplification Methods:
* _______ amplification
* _______ amplification
* _______ amplification
3 categories of Amplification Methods:
* Target amplification
* Probe amplification
* Signal amplification
TARGET AMPLIFICATION METHOD
Polymerase chain reaction (PCR)
* Discovered in ____ by ___________
* Escherichia coli plasmid ________
* 1st to be amplified
* Method was called “__________________________________________”
* User ________, more automated and more amenable to use.
* Any region of _____ can be chosen
* ___________ - copy of the target DNA / __________ of PCR
TARGET AMPLIFICATION METHOD
Polymerase chain reaction (PCR)
* Discovered in 1983 by Kary Mullis
* Escherichia coli plasmid pbr322
* 1st to be amplified
* Method was called “Polymerase-catalyzed chain reaction”
* User friendly, more automated and more amenable to use.
* Any region of DNA can be chosen
* Amplicons - copy of the target DNA / products of PCR
________________
* In a certain high temperature (_________) it will make the DNA to __________.
* The two strands of the target DNA will be __________.
* Sample dsDNA is denatured into ___ single strands
* Heating at _____ to _____ for several seconds to several minutes (__sec-__sec), depending on the template
Denaturing
* In a certain high temperature (94-95°c) it will make the DNA to separate.
* The two strands of the target DNA will be separated.
* Sample dsDNA is denatured into two single strands
* Heating at 94°c to 96°c for several seconds to several minutes (20sec-60sec), depending on the template
_____________
Derived from:
* Patient’s genomic DNA
* Patient’s _______________________
* DNA from: ________, _________, __________
* _______to 1ug of DNA is used
* Free from contaminating proteins
* No nicks/breaks
DNA template
Derived from:
* Patient’s genomic DNA
* Patient’s mitochondrial DNA
* DNA from: viruses, bacteria, parasites
* 100 ng to 1ug of DNA is used
* Free from contaminating proteins
* No nicks/breaks
Annealing
* Uses _______; serve as starting points to add the bases.
* The primers used are _________; oligonucleotides
* Binding of primers to the target.
* Primers used: 2 (_________, __________)
* Attachment or hybridization
* Two primers will prime the synthesis of DNA anneal (hybridize) to complementary sequences of the template.
* Two primers:
o Forward hybridize to __
o Reverse hybridizes to __
* Most ________ step
* ____to _____, __sec-__sec
Annealing
* Uses primers; serve as starting points to add the bases.
* The primers used are synthetic; oligonucleotides
* Binding of primers to the target.
* Primers used: 2 (forward, reversed)
* Attachment or hybridization
* Two primers will prime the synthesis of DNA anneal (hybridize) to complementary sequences of the template.
* Two primers:
o Forward hybridize to 5’
o Reverse hybridizes to 3’
* Most crucial step
* 50°c to 70°c, 20sec-90sec
Long primers: _________________ and hybridize at ________ rate
Short primers: ______________and hybridize at ________ rate
Long primers: more specific and hybridize at slower rate
Short primers: less specific and hybridize at faster rate
FACTORS THAT MAY AFFECT THE EXACT T
* ___________ conditions
* ______________________
* ___________ conditions
* __________________ (________ folding and hybridization between DNA strands)
FACTORS THAT MAY AFFECT THE EXACT T
* Reaction conditions
* Salt concentrations
* Template conditions
* Secondary structure (internal folding and hybridization between DNA strands)
_______________
___________ primer binding
Primers bind to ________________ sequences
__-__c and leads to formation of misprimes
Prevented by __________ PCR
Mispriming
Aberrant primer binding
Primers bind to unintended sequences
22-25c and leads to formation of misprimes
Prevented by hot-start PCR
__________________
_________ composed of two
Primers binding onto _____________
_________ the size of the utilized primer
Primer dimers
Artifact composed of two
Primers binding onto each other
Double the size of the utilized primer
_____________ or ______________
* The primers are extended using the ________________
* _________________: the enzyme capable of adding the bases.
* Primers and bases must be complementary with the target.
* __________________ occurs
* Addition of nucleotides (primer extension)
* __°c to __°c (optimal temp of the enzyme), __sec-__sec
* ____________ synthesizes a copy of the template DNA
* __________________ is used to add the bases; should be complementary with the template DNA
Extending or elongation
* The primers are extended using the DNA polymerase
* DNA polymerase: the enzyme capable of adding the bases.
* Primers and bases must be complementary with the target.
* DNA synthesis occurs
* Addition of nucleotides (primer extension)
* 68°c to 72°c (optimal temp of the enzyme), 10sec-60sec
* Polymerase synthesizes a copy of the template DNA
* DNA polymerase is used to add the bases; should be complementary with the template DNA
_________________________
Enzyme for the addition of nucleotides to the hybridized primers
High temperatures in denaturation step will ____________ the enzyme
DNA POLYMERASE
Enzyme for the addition of nucleotides to the hybridized primers
High temperatures in denaturation step will inactivate the enzyme
________________: mainly utilized in ______; from Thermus aquaticus; ____ loving: Thermostable; by ______________
_______________: utilized in __________________________ (_____): ____ serves as the starting material: uses transcriptase to facilitate the creation of RNA- from Thermus thermophilus
________________: Allows ___ o ___ to generate large products (_________ bases)
Taq polymerase: mainly utilized in PCR; from Thermus aquaticus; heat loving: Thermostable; by Randall Saiki
Tth polymerase: utilized in reverse-transcriptase PCR (RNA): RNA serves as the starting material: uses transcriptase to facilitate the creation of RNA- from Thermus thermophilus
Vent polymerase: Allows Taq o Tth to generate large products (30.000 bases)
MODIFIED Taq VERSIONS
____________________: 2x the half-life of Taq
In high temperatures; broader MgCl2 concentrations
For allele specific pcr and amplification of gc content
___________________________: incorporates ddNTPS
Chain ____________ sequencing
MODIFIED Taq VERSIONS
STOFFEL FRAGMENT: 2x the half-life of Taq
In high temperatures; broader MgCl2 concentrations
For allele specific pcr and amplification of gc content
THERMOSEQUENASE AND T7 SEQUENASE: incorporates ddNTPS
Chain termination sequencing
PCR REAGENTS
Typically, in a preformed solution called “_____________”
PCR REAGENTS
Typically, in a preformed solution called “master mix”
REAGENTS: COMPONENTS AND FUNCTIONS
_______ (oligodeoxynucleotides): Directs DNA synthesis to the desired region
___________________________: Building blocks that extend the primers
___: Monovalent cation (salt), for optimal hybridization of primers to template
___, pH ___: buffer to maintain optimal pH (_-___) for the enzyme reaction
_____________: divalent cation, required by the enzyme
____________: enzyme that extends the primers (adds DNTPS)
___-___ copies of template: sample DNA that being tested
REAGENTS: COMPONENTS AND FUNCTIONS
Primer (oligodeoxynucleotides): Directs DNA synthesis to the desired region
dATP, dCTP, dGTP,dTTP (dNTPs): Building blocks that extend the primers
KCI: Monovalent cation (salt), for optimal hybridization of primers to template
Tris, pH 8.4: buffer to maintain optimal pH (8-9.5) for the enzyme reaction
1.5 mM MgCl2: divalent cation, required by the enzyme
Polymerase: enzyme that extends the primers (adds DNTPS)
10²-10⁵ copies of template: sample DNA that being tested
___________________________
A mixture of four equimolar deoxynucleotide triphosphates: adenine (dATP); thymine (dTTP); guanine (dGTP); cytosine (dCTP); 0.1-0.5 mM concentrations of each
Concentration and purity may affect ___ results
_________________ (__-___ mM) are recommended for ______ to prevent breaking down to dNDP and dMNP = Inhibit ___
DEOXYNUCLEOTIDE BASES
A mixture of four equimolar deoxynucleotide triphosphates: adenine (dATP); thymine (dTTP); guanine (dGTP); cytosine (dCTP); 0.1-0.5 mM concentrations of each
Concentration and purity may affect pcr results
High concentrations (10-100 mM) are recommended for storage to prevent breaking down to dNDP and dMNP = Inhibit PCR
________________
* For high GC content
* Best for _________________
* Interferes with ____ staining
DEAZA DGTP
* For high GC content
* Best for autoradiography
* Interferes with EtBr staining
________________
* Provide optimal conditions for enzyme activity (pH _-___)
* Increased SAL concentration may denature ___________ slower than _______________
* Conditions vary with primers and templates
PCR BUFFERS
* Provide optimal conditions for enzyme activity (pH 8-9.5)
* Increased SAL concentration may denature long DNAs slower than shorter DNAs
* Conditions vary with primers and templates
_____ = may chelate Mg that will result to lower enzyme efficiency
EDTA = may chelate Mg that will result to lower enzyme efficiency
ACCESSORY COMPONENTES FOR BUFFERS
FOR OPTIMIZING REACTIONS
* _____________ (1%-10%)○ lower denaturing temperature
* _____________ (0.01 mm)
o reducer - enhance enzyme activity
* _______________ (10-100 ug/ml)
o binds inhibitors
o enzyme stabilizer
* ________________ (1%-10%)
o allows polymerase to access difficult areas
* Triton x-100
* Glycerol
* Dimethyl sulfoxide
ACCESSORY COMPONENTES FOR BUFFERS
FOR OPTIMIZING REACTIONS
* Formamide (1%-10%)○ lower denaturing temperature
* Dithiothreitol (0.01 mm)
o reducer - enhance enzyme activity
* Bovine serum albumin (10-100 ug/ml)
o binds inhibitors
o enzyme stabilizer
* Chaotropic agents (1%-10%)
o allows polymerase to access difficult areas
* Triton x-100
* Glycerol
* Dimethyl sulfoxide
_____________ and ____________
* first PCR system conducted
* replaced by automated methods and thermostable enzymes
Water baths and heat blocks
* first PCR system conducted
* replaced by automated methods and thermostable enzymes
_______________________________
* for rapid and automatically ramp to the required temperatures
* early version: _______ (vapor barriers) to prevent condensation of sample to the tube tops
Thermal cyclers/thermocyclers
* for rapid and automatically ramp to the required temperatures
* early version: Wax/oil (vapor barriers) to prevent condensation of sample to the tube tops
_____________ types:
* with ____________ detectors for measurement
* used in coronavirus
RT PCR types:
* with fluorescent detectors for measurement
* used in coronavirus
POSITIVE CONTROLS
* Ensures: _______ is active; _______ is optimal; ________ are priming right sequences; ____________ are cycling properly
NEGATIVE CONTROLS:
* Aka _____________________ or ______________
NEGATIVE TEMPLATE CONTROLS:
* With a dna that has no _____________; - there should be: - _____________
AMPLIFICATION CONTROLS:
* Contains a primer binding site; - determine _______________ (amplification failure)
POSITIVE CONTROLS
* Ensures: enzyme is active; buffer is optimal; primers are priming right sequences; thermal cyclers are cycling properly
NEGATIVE CONTROLS:
* Aka contamination control or reagent blank
NEGATIVE TEMPLATE CONTROLS:
* With a dna that has no target sequence; - there should be: - no annealing
AMPLIFICATION CONTROLS:
* Contains a primer binding site; - determine false negatives (amplification failure)
