Section 5 Flashcards
In vitro DNA amplification by PCR differs from in vivo DNA replication in that:
- PCR uses RNA primers
- PCR uses heat to separate the DNA double helix
- PCR uses ddNTPs
- PCR uses Mg2+ ions to coordinate reactants in the catalytic core of the polymerase
- PCR uses a heat-sensitive yeast DNA polymerase
PCR uses heat to separate the DNA double helix.
In PCR, the process of unwinding the DNA double helix, which is typically carried out by a helicase enzyme during in vivo DNA replication, is replaced with the application of heat. PCR still uses DNA primers and dNTPs, similar to in vivo replication. Both PCR and in vivo replication rely on Mg2+ ions to coordinate reactants in the catalytic core of the polymerase. Importantly, PCR is not heat-sensitive; in fact, it leverages heat to facilitate the separation of DNA strands, a key step in the reaction.
What are dideoxynucleotides and how are they used in Sanger sequencing?
Dideoxynucleotides, often abbreviated as ddNTPs, are chain-elongating inhibitors of DNA polymerase used in the Sanger method for DNA sequencing. In Sanger sequencing, ddNTPs lack the 3’-hydroxyl group needed for the next step in DNA synthesis. Their incorporation at specific points in the growing DNA strand terminates synthesis and helps determine the sequence of the target DNA.
Describe the steps involved in Sanger Sequencing.
- DNA Denaturation: The DNA sample is heated to denature the double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), forming a template strand and a complementary strand.
- Primer Annealing: A DNA primer is annealed to the template strand, providing a free 3’ hydroxyl group for DNA polymerase to add nucleotides.
- Free Nucleotides (dNTPs): Four reaction mixtures are set up, each containing the template strand with its primer, DNA polymerase, and free nucleotides (dNTPs).
- Modified Nucleotides (ddNTPs): Modified nucleotides called ddNTPs are added to each reaction mixture. Only one type of ddNTP (ddATP, ddTTP, ddCTP, or ddGTP) is added to each reaction mixture. ddNTPs are present at a much lower concentration than dNTPs, allowing for some extension of the synthesized strand with unmodified dNTPs before a ddNTP is incorporated.
- Chain Termination: During this step, chain-terminating ddNTPs are used. These ddNTPs lack a 3’-OH group necessary for the formation of a phosphodiester bond between nucleotides, causing DNA polymerase to stop extending the DNA strand when a ddNTP is added.
- Gel Electrophoresis: A sample is collected from the reaction mixtures and subjected to gel electrophoresis, separating DNA fragments by size. Each reaction mixture is added to a separate lane on the gel. The radiolabeled primer is detected through autoradiography.
The result of gel electrophoresis displays all possible chain lengths separated by one nucleotide. Shorter fragments travel further on the gel than longer ones, allowing the determination of the DNA sequence complementary to the template strand.
Why is polyacrylamide gel used rather than agarose gel in Sanger sequencing?
Because of its high resolving power, and it can separate DNA strands that differ in length by 1 base pair
What sequence of DNA does Sanger sequencing provide upon analyzation?
The complementary sequence
How can you optimize PCR conditions for more selective amplification of your desired product?
- Increase annealing temperature
- Reduce salt (influences stability)
- increasing [MgCl2] = less stringent conditions
- decreasing [MgCl2] = sub-optimal polymerase activity
What does stringent mean?
In PCR, “stringent conditions” refer to the conditions that promote the binding (annealing) of primers to their target DNA sequences with high specificity. High stringency conditions typically involve using higher annealing temperatures or lower salt concentrations to ensure that the primers only bind to sequences that closely match their target, reducing non-specific interactions.
In a broader sense, “stringent” can be used to describe any situation where strict criteria or high standards are applied to achieve a specific outcome.
How can you improve the selectivity of your desired DNA amplification in PCR?
By increasing the annealing temperature during PCR.
Explanation: In PCR, the annealing step involves binding primers to the DNA template. By increasing the annealing temperature, you make it more specific, so primers will only bind to sequences that closely match your target, increasing the selectivity of the amplification.
How does adjusting salt concentration affect PCR conditions?
Modifying the salt concentration in PCR can impact reaction conditions. Increasing the concentration of MgCl2 makes the conditions less stringent, while reducing MgCl2 results in sub-optimal polymerase activity.
Explanation: In PCR, salt concentration, often represented by MgCl2, influences the reaction conditions. Increasing MgCl2 concentration makes the conditions less stringent, meaning it allows for more relaxed primer binding, potentially leading to non-specific amplification. On the other hand, reducing MgCl2 can impair the polymerase’s activity, affecting the success of the reaction.
How can you obtain enough copies of a purified DNA segment for sequencing?
Two common methods used in the lab are in vitro PCR amplification and in vivo DNA replication through molecular cloning.
What is in vitro PCR amplification used for in DNA sequencing?
In vitro PCR amplification is used to obtain sufficient copies of a DNA segment with known flanking sequences, enabling targeted amplification for sequencing
How is in vitro PCR amplification adapted for DNA segments with completely unknown sequences?
For DNA segments with unknown sequences, synthetic adapters with known sequences can be ligated to the ends to serve as primer binding regions for PCR amplification.
What is the purpose of in vivo DNA replication in the context of DNA sequencing?
In vivo DNA replication, achieved through molecular cloning, is used to amplify a DNA segment of interest by incorporating it into a vector and replicating it within bacteria, allowing the production of sufficient DNA for sequencing.
What is ligation?
The enzymatic joining of two nucleic acid fragments
Describe the difference between In vivo vs in vitro DNA amplification.
In VIVO, In VITRO
Genomic or plasmid DNA, DNA containing segment to be amplified
Primase, pair of target-specific DNA primers
DNA Pol III, Thermostable Taq polymerase
Helicase, Denature DNA by heat
dNTPs, dNTPs
What is molecular cloning, and how does it provide large quantities of purified DNA for sequencing?
Molecular cloning is the process of isolating and generating recombinant DNA molecules. These recombinant DNA molecules are placed in host organisms for replication and study. In DNA cloning, a specific gene or DNA segment is separated from a larger chromosome and incorporated into a small carrier DNA molecule. This modified DNA is then introduced into a host cell, leading to its replication. This process increases both the cell number and the copy number of the cloned DNA in each cell, thereby providing large quantities of purified DNA for sequencing.
What are some practical applications of molecular cloning besides DNA amplification for sequencing?
Molecular cloning has various applications besides DNA amplification. It can be used to express a protein of interest in host cells, allowing researchers to study its function by forcing the cloned DNA to be translated into protein in the cells. Additionally, molecular cloning can be used to create mutant forms of proteins or produce tagged versions for visualization purposes. While molecular cloning has multiple applications, in this course, the primary focus is on its ability to amplify DNA for sequencing applications.
What are the five general steps involved in molecular cloning?
- Obtaining the DNA segment to be cloned.
- Selecting an appropriate carrier molecule of DNA capable of self-replication (cloning vectors).
- Joining two DNA fragments covalently with the enzyme DNA ligase, creating recombinant DNA.
- Moving recombinant DNA from the test tube to a host organism.
- Selecting or identifying host cells that contain recombinant DNA.
How is the DNA obtained in molecular cloning?
Enzymes called restriction endonucleases are often used to cleave genomic DNA into smaller fragments suitable for cloning. Alternatively, genomic DNA may be sheared randomly into fragments, or some DNA segments to be cloned are synthesized.
What is the role of cloning vectors in molecular cloning, and can you provide examples of such vectors?
Cloning vectors are small DNA molecules capable of self-replication, acting as carriers for new DNA. Examples of cloning vectors include plasmid vectors and bacterial artificial chromosomes (BACs).
What is recombinant DNA, and how is it created in molecular cloning?
Recombinant DNA is formed by covalently linking segments from two or more sources. In molecular cloning, recombinant DNA is created by joining the cloning vector to the DNA fragment to be cloned using the enzyme DNA ligase.
What is the purpose of moving recombinant DNA from the test tube to a host organism in molecular cloning?
Moving recombinant DNA to a host organism allows for DNA replication. Host organisms, often bacteria, provide the enzymatic machinery for DNA replication, enabling the production of multiple copies of the recombinant DNA.
How are host cells containing recombinant DNA selected or identified in molecular cloning?
Host cells containing recombinant DNA are selected or identified using features of the cloning vector that allow the host cells to survive in a specific environment. For example, antibiotic resistance genes are often included in the vector, making cells with the vector “selectable” in the presence of antibiotics. The propagation (cloning) of these transformed cells results in many copies of the recombinant DNA.
What are plasmids, and why are they useful in molecular cloning?
Plasmids are circular DNA molecules found in bacteria that replicate separately from the bacterial chromosome. They are useful for cloning DNA fragments that are less than approximately 15,000 base pairs in length. Plasmids contain specialized sequences that enable them to use the host cell’s resources for their own replication and gene expression. These properties are valuable to researchers who can engineer plasmids as vectors for cloning specific DNA segments. Natural plasmids often have a symbiotic role in the cell, providing genes that confer resistance to antibiotics or perform new functions for the host cell.
Describe the features of a plasmid
Ori: The plasmid pBR322 has an origin of replication (ori) - a sequence where replication is initiated by cellular enzymes. This sequence is required to propagate the plasmid.
Restriction Sequences: Several unique restriction sequences in pBR322 are targets for restriction endonucleases (PstI, EcoRI, BamHI, SalI, and PvuII), providing sites where the plasmid can be cut to insert foreign DNA.
Number of Base Pairs: The small size of the plasmid (4,361 bp) facilitates both its entry into cells and the biochemical manipulation of the DNA. This small size is generated by trimming away unnecessary DNA segments from a larger parent plasmid.
Antibiotic Resistance: The plasmid contains genes that confer resistance to the antibiotics tetracycline and ampicillin. This resistance allows the selection of cells that contain the intact plasmid or a recombinant version of the plasmid using these antibiotics.
How does Sanger Sequencing work in terms of DNA synthesis?
Sanger Sequencing relies on the enzymatic synthesis of a complementary DNA strand to the one being analyzed, using a labeled primer and dideoxynucleotides.
What is the role of dideoxynucleotides (ddNTPs) in the Sanger sequencing reaction?
In Sanger Sequencing, dideoxynucleotides (ddNTPs) are nucleotide analogs that lack the 3’-hydroxyl group required for the next step in DNA synthesis, leading to chain termination.
What is the key difference between deoxynucleotides and dideoxynucleotides?
Dideoxynucleotides, unlike deoxynucleotides, have H atoms at both the 2’ and 3’ positions, making them chain-elongating inhibitors of DNA polymerase, which are used in the Sanger method for DNA sequencing.
True or false:
In sanger sequencing, newly synthesized strands will terminate at the incorporation of an A when we add ddATP, the incorporation of a C if we add ddCTP, a G when we add ddGTP and a T when we add ddTTP
True
What are the differences between Sanger sequencing and dye-terminator Sanger sequencing?
In dye-terminator Sanger sequencing, all four dideoxynucleotides (ddNTPs) are added to the same reaction mixture, and each type is labeled with a distinct fluorescent dye (e.g., ddCTP in blue, ddATP in green, ddGTP in yellow, ddTTP in red).
In dye-terminator Sanger sequencing, the products of different sizes are separated using capillary electrophoresis. Here, the fluorescently labeled segments are excited by a laser, and the emitted wavelength (red, green, yellow, or blue) is detected one nucleotide at a time. This method offers advantages such as avoiding radioactivity, high throughput, and the ability to sequence longer DNA fragments, with a maximum read length of 1000-1500 base pairs.
How are host cells containing recombinant DNA selected or identified in molecular cloning?
Cloning vectors typically have features that allow host cells containing them to survive in an environment where cells lacking the vector would die, such as antibiotic resistance. This makes cells with the vector “selectable.” The transformed cells can be propagated to produce many copies of the recombinant DNA.
What is the role of the host organism in molecular cloning, and what is commonly used as the host?
The host organism provides the enzymatic machinery for DNA replication. Bacteria are often used as the host organism for molecular cloning.
How are two DNA fragments covalently joined in molecular cloning?
The enzyme DNA ligase is used to link the cloning vector to the DNA fragment to be cloned, resulting in the formation of recombinant DNA.
