Working with RNA/DNA Flashcards
What is reverse transcription?
Reverse transcription is a process by which RNA molecules are reverse transcribed into complementary DNA (cDNA) molecules. This process is catalyzed by an enzyme called reverse transcriptase.
What are the components required for reverse transcription?
1.RNA template: The RNA molecule that will serve as a template for the synthesis of cDNA.
2. Reverse transcriptase enzyme: This enzyme catalyzes the synthesis of cDNA from the RNA template.
3. Primers: Short sequences of single-stranded DNA or RNA that serve as starting points for the reverse transcriptase enzyme to begin cDNA synthesis.
4. Nucleotides: The building blocks of DNA and RNA, which are used by the reverse transcriptase enzyme to build the cDNA strand.
5. Buffer solution: A solution that provides the optimal conditions for the reverse transcription reaction to occur, such as the appropriate pH, salt concentration, and cofactors.
6. MgCl2: A cofactor that is often included in the reverse transcription reaction to enhance the activity of the reverse transcriptase enzyme.
7. RNase inhibitor: An enzyme inhibitor that is often added to the reverse transcription reaction to prevent degradation of the RNA template by RNases, which are enzymes that degrade RNA.
What is MMLV transcriptase?
Moloney Murine Leukemia Virus reverse transcriptase is a type of reverse transcriptase enzyme. DNA polymerase that catalyzes the synthesis of a complementary DNA (cDNA) strand from a single-stranded RNA template. It requires a primer to initiate cDNA synthesis, and uses deoxynucleoside triphosphates (dNTPs) as building blocks to add nucleotides to the growing cDNA strand.
What are the limitations of MMLV?
- MMLV transcriptase may not efficiently transcribe RNA templates that have complex secondary structures or strong RNA-RNA interactions. This can lead to incomplete or inaccurate cDNA synthesis, as the enzyme may not be able to access certain regions of the RNA template.
- MMLV transcriptase may have difficulty synthesizing cDNA from long RNA templates, as it can become less processive and less efficient with increasing template length. This can result in incomplete cDNA synthesis or low yields of cDNA.
- MMLV transcriptase is sensitive to RNase contamination, which can degrade the RNA template and compromise cDNA synthesis. Careful handling and storage of the enzyme and reagents is necessary to minimize the risk of RNase contamination.
Why is superscript III transcriptase good?
- has a high processivity, which means that it can efficiently synthesize long cDNA strands. This is important for applications such as full-length cDNA library construction, where it is necessary to capture the entire coding sequence of a gene.
- has a high fidelity, which means that it has a low error rate during cDNA synthesis. This is important for applications such as cloning or expression analysis, where accurate representation of the RNA template is critical.
- wide range of temperatures, from 42°C to 55°C, which makes it useful for applications such as RT-PCR, where high temperatures are required for the amplification step.
What are the common types of primers used in reverse transcription?
- Oligo-dT primers are the most commonly used primers for reverse transcription. These primers are short, synthetic DNA sequences that anneal to the poly(A) tail of mRNA templates, allowing the reverse transcriptase to initiate cDNA synthesis at the 3’ end of the RNA molecule. Oligo-dT primers are suitable for most applications and are typically used in combination with random primers.
- Random primers are short, synthetic DNA sequences that contain a mix of all four nucleotides at their 3’ end. These primers anneal randomly along the RNA template, allowing the reverse transcriptase to initiate cDNA synthesis at multiple sites along the RNA molecule. Random primers are useful for generating cDNA libraries, as they can capture a broader range of RNA species.
- Gene-specific primers are designed to anneal to a specific RNA sequence of interest, such as a target gene or a region of interest within a larger transcript. These primers are useful for applications such as quantitative PCR, where specific amplification of a target RNA sequence is required.
What is wrong with using oligo-dt primers?
- Oligo-dT primers can bias cDNA synthesis towards the 3’ end of the mRNA template, which can result in incomplete coverage of the target transcript. This can be particularly problematic for long transcripts or transcripts with complex secondary structure.
- Oligo-dT primers can have difficulty annealing to short or degraded mRNA templates, leading to incomplete cDNA synthesis.
- Oligo-dT primers can misprime at non-poly(A) regions of the mRNA template, resulting in the synthesis of non-specific cDNA.
- The poly(A) tail of mRNA transcripts can vary in length, and oligo-dT primers may not anneal equally well to transcripts with shorter or longer poly(A) tails.
Why are random primers used?
- Capture of non-polyadenylated RNA species: Some RNA species, such as non-coding RNA or viral RNA, may lack a poly(A) tail and cannot be captured by oligo-dT primers. Random primers allow for the capture of these non-polyadenylated RNA species, providing a more comprehensive representation of the transcriptome.
- Coverage of the 5’ end of transcripts: Oligo-dT primers can bias cDNA synthesis towards the 3’ end of the mRNA transcript, making it difficult to capture the 5’ end of the transcript.
- Overcoming issues with RNA quality: RNA degradation or fragmentation can make it difficult for oligo-dT primers to anneal to the poly(A) tail of mRNA transcripts, resulting in incomplete cDNA synthesis. Random primers can anneal to any region of the RNA template, providing an alternative priming strategy that can overcome these issues.
What are gene specific primers?
Gene-specific primers are short DNA sequences that are designed to anneal to a specific target sequence within a mRNA transcript. Issues is that there is lower yield due to specific transcript.
What is PCR and what is it used for?
PCR (polymerase chain reaction) is a laboratory technique that allows for the amplification of a specific DNA sequence or target region. Its used for amplifying specific genes or DNA sequences for cloning or analysis
Identifying genetic mutations or variations
Quantifying gene expression levels using qPCR
What are the steps of PCR?
The PCR process involves three basic steps: denaturation, annealing, and extension. During denaturation, the double-stranded DNA template is heated to separate the two strands. Then, during annealing, short primers (complementary DNA sequences that flank the target region) are added to the mixture, and they anneal to the single-stranded DNA template. Finally, during extension, a heat-stable DNA polymerase enzyme copies the target sequence using the primers as a starting point, producing two copies of the target sequence.
What are the components required for PCR reaction?
- DNA template: The DNA template contains the sequence to be amplified. This can be genomic DNA, plasmid DNA, cDNA, or any other source of DNA.
- Primers: Two primers are required, one for each end of the target DNA sequence. The primers are typically short (18-25 nucleotides) and specific to the target sequence, and they are used to define the boundaries of the DNA to be amplified.
- Taq polymerase: A DNA polymerase enzyme is required to synthesize new DNA strands. Taq polymerase is a commonly used enzyme for PCR, derived from the bacterium Thermus aquaticus. It is heat-stable and can withstand the high temperatures used during PCR.
- Deoxynucleoside triphosphates (dNTPs): The four building blocks of DNA (dATP, dCTP, dGTP, and dTTP) are required for DNA synthesis. They are added in excess to ensure that the polymerase has an ample supply of building blocks.
- Buffer: The buffer provides the necessary pH and salt concentration for the reaction to occur. It also contains other components that can affect the efficiency and specificity of the reaction.
- Water: Water is used to dilute the other components and to bring the reaction to the desired final volume.
How many copies of target DNA can be obtained within 4 hours from a single DNA molecule
1 billion copies
What is gel electrophoresis?
Gel electrophoresis is a laboratory technique used to separate and analyze DNA, RNA, and proteins based on their size, charge, and other physical properties.
What is the general protocol to make a 1% Agarose gel?
To make a 100 ml gel, you would need 1 g of agarose powder and 100 ml of buffer.
1. Add the agarose powder to a clean, heat-resistant flask or beaker.
2. Add the buffer to the flask or beaker and stir to dissolve the agarose powder.
3. Heat the mixture on a microwave or hot plate until the agarose is completely dissolved. Avoid overheating the agarose solution, as this can cause degradation of the agarose and affect the quality of the gel.
4. Let the agarose solution cool for a few minutes until it is still liquid but not too hot to touch.
5. If desired, add ethidium bromide to the solution at a concentration of 0.5 µg/ml and mix gently.
6. Pour the agarose solution into the gel casting tray and insert the comb at the desired position to create wells.
7. Allow the gel to solidify for about 30-60 minutes at room temperature.
8. Once the gel is solid, carefully remove the comb and place the gel in the electrophoresis chamber. Fill the chamber with the appropriate buffer.
