36 Questions
What is gene amplification?
An increase in the copy of a particular gene without an increase in the number of copies of other genes
How can gene amplification occur naturally?
Through gene duplication, replication and repair slippage, ectopic recombination, aneuploidy, and polyploidy
What is the term for the piece of DNA or the gene that gets amplified naturally or artificially?
Amplicon
How is gene amplification artificially done in a lab?
By polymerase chain reaction (PCR)
What is the recommended range for GC content in primer sequences?
40-60%
Why is it important to check primer specificity by BLAST?
To ensure the primers only bind to the target sequence
What is the recommended length range for primer sequences?
18-25 bp
What is the formula to calculate the melting temperature (Tm) of a primer?
Tm = (G + C) x 4 + (A + T) x 2
What does the annealing temperature (Ta) depend directly on?
Length and GC composition of the primers
What does the presence of a GC clamp in a primer sequence refer to?
Having G or C bases within the last five bases from the 5' end of the primer
What is the recommended range for melting temperatures (Tm) of primer sequences?
50-60 °C
Why is it important to know the product size of the amplified target sequence?
To ensure the target sequence is amplified and of the expected size
Which technique utilizes RNA intermediates for gene amplification?
Transcription-mediated amplification (TMA)
What is a specific advantage of Ligase chain reaction (LCR) over PCR?
Increased specificity for point mutations
Which method is commonly used to produce billions of copies of a target DNA sequence in a short time?
Polymerase chain reaction (PCR)
What governs the three distinct steps of denaturation, annealing, and extension in PCR?
Temperature
What determines the number of copies produced in PCR?
Number of cycles
What is a key component required for PCR to target the DNA to be amplified?
Synthetic oligonucleotide primers
What is the role of reverse transcriptase in Transcription-mediated amplification (TMA)?
Catalyzes RNA to DNA conversion
What is the primary purpose of post-PCR analysis?
Primer design and PCR optimization
What is the main advantage of Ligase chain reaction (LCR) over PCR?
Increased specificity for point mutations
What is the primary function of Ligase chain reaction (LCR)?
Detection of point mutations
Which enzyme is essential for the rapid amplification of target genes in Transcription-mediated amplification (TMA)?
RNA polymerase
What is the main role of DNA polymerase in PCR?
Synthesizes new DNA strands
What is the main purpose of gene amplification?
Disease diagnosis and understanding disease progression
Which gene amplification technique is more specific than PCR and used to identify point mutations?
LCR
What is the main role of transcription-mediated amplification (TMA)?
Rapidly amplifying target genes
What is the main function of PCR in the lab?
Producing multiple copies of a desired gene
What governs the three distinct steps of PCR: denaturation, annealing, and extension?
Temperature
How many copies of the target DNA can PCR produce in about 20 cycles?
About a million
What is the main requirement for PCR to target the DNA to be amplified?
Specific DNA sequence information
What is the mechanism of PCR primarily involved in?
Denaturation of template DNA, primer annealing, and DNA synthesis
What does post-PCR analysis involve?
Primer design, primer specificity, PCR optimization, and visualization of results
What determines the number of copies produced in PCR?
2n, where n is the number of cycles
What is the main role of ligase chain reaction (LCR) in gene amplification?
Identifying point mutations
What is the primary function of gene amplification in research and diagnosis?
To produce desired gene products
Study Notes
Gene Amplification Techniques: PCR, LCR, and TMA
- Retrotransposition is a type of transposon that copies itself through RNA intermediates, and is commonly found in tumour cells.
- Amplification of genes is useful for disease diagnosis, understanding disease progression, and can lead to drug resistance in cancer cells.
- Artificial gene amplification is used in research, diagnosis, recombinant DNA technology, and to produce desired gene products.
- Polymerase chain reaction (PCR) is a commonly used method to produce multiple copies of a desired gene in the lab, and is essential in modern science and biotechnology.
- Ligase chain reaction (LCR) is another method of gene amplification, more specific than PCR, and widely used to ascertain point mutations such as in genetic diseases.
- Transcription-mediated amplification (TMA) utilizes RNA polymerase and reverse transcriptase to rapidly amplify target genes.
- PCR is a means to amplify a particular piece of DNA and can make billions of copies of a target sequence of DNA in a short time.
- PCR requires specific DNA sequence information to target the DNA to be amplified and uses synthetic oligonucleotide primers.
- PCR proceeds in three distinct steps governed by temperature: denaturation, annealing, and extension.
- The mechanism of PCR involves denaturation of template DNA, primer annealing, and DNA synthesis by DNA polymerase.
- PCR can produce about a million copies of the target in only 20 cycles, and the number of copies is determined by 2n, where n is the number of cycles.
- Post-PCR analysis involves primer design, primer specificity, PCR optimization, and visualization of results using agarose gel electrophoresis.
Explore gene amplification techniques like PCR, LCR, and TMA used in research, diagnosis, and biotechnology. Learn about the mechanisms, applications, and significance of amplifying genes in disease diagnosis and drug resistance.
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