Polymerase Chain Reaction Flashcards
A powerful method for amplifying particular segments of DNA.
PCR
PCR is carried out entirely biochemically in ____.
Vitro
PCR uses this enzyme to direct the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template
DNA polymerase
DNA polymerase adds nucleotides to the _____ of a custom-designed oligonucleotide when it is annealed to a longer template DNA
3` end
The sample DNA that contains the target sequence.
DNA template
Type of enzyme that synthesizes new strands of DNA complementary to the target sequence
DNA polymerase
DNA polymerase that is most commonly used in PCR
Taq DNA polymerase
Enzyme that is used widely because of its higher fidelity when copying DNA
Pfu DNA polymerase
Two capabilities that makes DNA polymerase suitable for PCR
1) they can generate new strands of DNA using a DNA template and primers;
2) they are heat resistant.
Short pieces of single-stranded DNA that are complementary to the target sequence
Primers
“building blocks” for new DNA strands
Nucleotides (dNTPs or deoxynucleotide triphosphates)
What happens in Denaturation
DNA template is heated to 94°C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.
What happens in Annealing
The mixture is cooled to anywhere from 50-70°C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.
What happens in Extension
The reaction is then heated to 72°C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.
GIve 5 applications of PCR
- Disease diagnosis and detection of pathogens
- Used in forensics laboratories
- Used to identify and to explore relationships among species in the field of evolutionary biology.
- In anthropology, it is used to understand the ancient human migration patterns.
- In archaeology, it has been used to spot the ancient human race.
Give 5 types of PCR
- Conventional PCR
- Real-time PCR
- Quantitative real time PCR (Q-RT PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Multiplex PCR
- Nested PCR
Conventional PCR is applied in
- Selective DNA isolation
- Amplification and quantification of DNA
- Medical and diagnostic approaches
- Infectious disease diagnosis
- Forensic studies
PCR-based technique that couples amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction
Quantitative PCR
A time-consuming process where the PCR products are analysed through gel electrophoresis.
Conventional PCR
qPCR facilitates the analysis of conventional PCR by ____________.
Providing real time detection of products during the exponential phase.
The principle of real-time PCR depends on the use of
Fluorescent dyes
Applications of Quantitative PCR
- Genotyping and quantification of pathogens
- MicroRNA analysis
- Cancer detection
- GMOs detection
- Microbial load testing
Real-time PCR is commonly used to measure ___________.
Gene expression
It is best suited for studies of small subsets of genes.
Real-time PCR
It can only be used for studying known genes.
Real-time PCR
First step in a real-time PCR reaction
The conversion of RNA to complementary DNA (cDNA)
The conversion of RNA to complementary DNA (cDNA) is called
Reverse transcription
Second step in Real Time PCR
Using fluorescent reporters and a PCR reaction to amplify and detect specific genes.
Two types of fluorescent reporters that are commonly used
SYBR green and Taqman probes
Is a dye that fluoresces only when bound to double stranded DNA
SYBR green
This fluorescent are made of a gene-specific nucleic acid probe, joined to reporter and quencher molecules
Taqman probes
This probe binds to the DNA between the forward and reverse primer.
Taqman probes
In a Taqman probe, it absorbs the fluorescence emitted by the reporter
Quencher molecule
The advantage of the Taqman method is that _____________.
Probes with different coloured reporters can be combined in multiplex assays.
For both SYBR green and Taqman methods, the amount of fluorescence in a sample is detected in ________ and _________.
‘Real-time’ and plotted against the cycle number
The amount of fluorescence is ________ to the amount of ___________ .
proportional, PCR product
The design of real-time PCR experiments requires ____________.
Prior knowledge of the gene sequence
It is a modification of conventional PCR, whereby RNA molecules are first converted into complementary DNA(cDNA) molecules that can then be amplified by PCR.
Reverse transcription PCR
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First step in RT-PCR
The RNA template is first converted into a complementary DNA (cDNA) using reverse transcriptase.
The cDNA then acts as a template for exponential amplification using PCR
RT-PCR can be conducted either in ________ or _________.
Either in a single tube or as two steps in different tubes
The one-step method in RT-PCR is ____________ and ________________ .
more effective with fewer chances of contamination, incorporation of variations
Applications of Reverse Transcriptase PCR
- Research methods
- Gene insertion
- Genetic disease diagnosis
- Cancer detection.
RT-PCR is commonly associated with q-PCR forming
Reverse Transcriptase Real-Time PCR
Allows quantification of DNA in real-time after the amplification.
Reverse Transcriptase Real-Time PCR
A common molecular biology technique used for the amplification of multiple targets in a single PCR test run.
Multiplex PCR
What are used for the amplification of DNA in a thermal cycler in Multiplex PCR.
Multiple primers and A temperature-mediated DNA polymerase
Why are all the primer’s pairs designed for Multiplex PCR, have to be optimized
So that the same annealing temperature is optimal for all the pairs during PCR.
Applications of Multiplex PCR
- Genotyping
- Detection of pathogens or genetically modified organisms
- Mutation and polymorphism analysis
- Microsatellite STR analysis
How is Multiplex PCR used in diagnostic laboratories
It is useful in detecting different microorganisms that cause the same types of diseases.
Give advantages of Multiplex PCR
- Cost effective — fewer dNTPs, enzymes, and other consumables
- Fewer pipetting errors
- Higher throughput
- Time saving
- More information with less sample
- More data from limited starting materials
- Less input material required
Considerations of Multiplex PCR
- Time and cost of optimization and validation
- Primer design for some multiplexed targets can be complex
- Some prior knowledge of the relative quantities of the target sequences is advantageous for obtaining accurate results
Is a useful modification of PCR technology where the sensitivity and specificity of the reaction is enhanced by preventing the non-specific binding with the help of the two sets of primers
Nested PCR
What happens to the first set of primers in Nested PCR
The first set of primer binds outside of our target DNA and amplifies larger fragment
What happens to the second set of primers in Nested PCR
The second set of primer amplifies only the target DNA/the other set of primer binds specifically at the target site.
Is a helpful method for the phylogenetic studies and detection of different pathogens
Nested PCR
This PCR technique has higher sensitivity
Nested PCR
Nested PCR has higher sensitivity; hence even if the sample contains lower DNA _____________.
It can be amplified which is not feasible in the conventional PCR technique.
A method in which, where identification of DNA of interest inserted into the plasmid is obtained by designing the inserted DNA specific primers
Colony PCR
The bacterial colony containing the plasmid can directly be amplified using __________.
Two sets of primers
What is the first set of primer in Colony PCR
The first set is of the insert specific primers which amplify the insertion sequence
What is the second set of primer in Colony PCR
The second set is of vector-specific flanking primers, which amplifies the plasmid DNA other than the inserted DNA
A bacterial colony is taken and ________.
Added directly into the master mix containing all other PCR reagents
Main application of Colony PCR
Is in the identification of correct ligation and insertion of inserted DNA into bacteria as well as yeast plasmid.
Is one of the many variants of the polymerase chain reaction that is used to amplify DNA when only one sequence is known
Inverse PCR
Inverse PCR allows ______________.
Amplification to be carried out even if only one sequence is available from which primers may be designed
What is Inverse PCR used for
For the determination of insert locations of various transposons and retroviruses in the host DNA.
What is involved in Inverse PCR
A series of restriction digestion followed by ligation, resulting in a a looped fragment that can then be primed for PCR through a single section of known sequence.
