DNA synthesis in the Lab Flashcards
Polymerase Chain Reaction (PCR)
1. Denaturation
==> Mixture is heated high temperature to separate DNA strands
-> DNA strands held together by weak
forces, e.g., H-bonds == Can be separated (melted) by heating
2. Annealing
==> As the reaction cools down, the primers anneal to the specific target sequences on the separated strands.
=> Cooled to allow specific primers anneal (H bond) to where you want to amplify
3. Extension
=> A heat-stable DNA polymerase extends the primers, synthesizing new strands of DNA. Polymerase catalyses formation of phosphodiester bond, adding nucleotide to growing chain
4. Amplification: The newly synthesized DNA, or amplicon, serves as the template for the next cycle, leading to the doubling of the target sequence with each cycle.
Which of the following would increase Tm?
A. Increased % of GC
B. Increased length
C. Increased ionic strength
D. Increased pH (> 9)
E. No idea
Tm = the temperature it takes to separate out your DNA strands
G-C would increase : more H bonds
Increased length : more H bonds
ionic strength will increase : positive ions will reduce the replusion of the negatively charged backbone and harder to separate
pH increased will decrease Tm : proton acceptors and will deprotonate the hydrogren bonds between the bases = disrupt base pairing
primers
In lab processes like PCR, we are typically only interested in copying a specific part of the DNA
== DNA polymerases can only add nucleotides to an existing chain; they cannot start a new chain from scratch. Therefore, they require a primer - a short sequence of nucleotides - to which they can add the rest of the nucleotides.
We can design primers to only amplify what we are interested in: By designing primers that bind to specific parts of the DNA, we can control where the DNA polymerase starts and ends, and thus which part of the DNA gets copied.
Need to be specific – ~20 nt is usually good: The primers need to bind to the DNA at exactly the right place, so they need to be very specific. A length of around 20 nucleotides usually provides enough specificity to bind to one unique location in the genome.
The primers always have the 3’ OH (hydroxyl) group at their 3’ end. The DNA polymerase will add nucleotides to this end, thus moving in the 5’ to 3’ direction along the template strand of the DNA.
primer design for PCR
- The Tm of your primers should be similar so they can bind to the template DNA at the same temperature.
–> Appropriate Tm, and similar for forward and reverse primers: Tm refers to the “melting temperature” of a DNA strand, or the temperature at which 50% of the DNA strands in a sample are separated or “melted”. - Primers should be designed so that they don’t form secondary structures by binding to themselves, as this can interfere with their ability to bind to the target sequence on the DNA template
==> Doesn’t anneal to itself: - If the forward and reverse primers bind to each other, they won’t be available to bind to the template DNA. However, there are occasions where this is intentionally done, such as when generating mutations in the DNA.
==> Don’t anneal to each other: - Sequences with a higher GC content will have a higher Tm because GC pairs form three hydrogen bonds (compared to two for AT pairs), making them more stable.
The text also mentions the importance of being careful with primer design even when introducing intentional mutations, to ensure that the primers can still bind to the template DNA. It also notes that the ionic strength and annealing temperature can influence how specifically your primers bind.
In some applications, primers might need to have a specific GC% or end with G or C to ensure more stable binding.
role of Mg2+
Required for DNA polymerase: Mg2+ ions are needed for the function of DNA polymerase. They facilitate the reaction where DNA polymerase adds nucleotides to the growing DNA strand.
DNA has a negative charge due to the phosphate groups in its backbone. Mg2+ ions, being positively charged, can shield these negative charges. If there’s too much Mg2+, it can bring the DNA strands closer together by reducing the repulsion between them, thus promoting base pairing.
==> Too much will shield the negative phosphates, promoting base pairing:
Consequences:
* Increased Tm: More Mg2+ can increase this temperature because it promotes base pairing, making the DNA strands stick together more strongly.
* Reduces specificity of primer binding: When Mg2+ promotes base pairing, it may cause the primers to bind less specifically. In other words, they might bind to sequences that are not perfectly complementary, which can lead to errors in the DNA synthesis.
role of Taq Polymerase
Taq polymerase is derived from a bacterium called Thermus aquaticus, which was discovered in hot springs.
The bacterium Thermus aquaticus is able to survive in the high temperatures of hot springs because it produces heat-stable enzymes that do not denature, or lose their function, when exposed to heat.
==> Still works after heating to 95°C!
Works optimally at 72°C – improves specificity: optimal temperature for Taq polymerase to function.
Thermus aquaticus discovered in hot springs: Taq polymerase is derived from a bacterium called Thermus aquaticus, which was discovered in hot springs.
Has some heat stable enzymes, including a DNA polymerase: The bacterium Thermus aquaticus is able to survive in the high temperatures of hot springs because it produces heat-stable enzymes that do not denature, or lose their function, when exposed to heat. One of these enzymes is Taq DNA polymerase.
Still works after heating to 95°C!: One of the key steps in PCR is heating the reaction to 95°C to separate, or “denature,” the DNA strands. Taq polymerase is able to survive this step and still function afterwards, which makes it ideal for PCR.
Works optimally at 72°C – improves specificity: The temperature is then lowered to about 72°C for the extension step, where Taq polymerase adds nucleotides to the primers to create the new DNA strands. This is the optimal temperature for Taq polymerase to function.
Benefits
* Speedy and saves adding polymerase each cycle!
==> efficient (speedy) and convenient as there is no need to add more polymerase in each cycle of the PCR, likely because it is heat-stable and can survive multiple rounds of heating and cooling
Electrophoresis
The phosphate groups in the DNA backbone carry a negative charge.
The negative charge of the DNA molecule, relative to its size (i.e., the number of nucleotides), is constant. This allows DNA fragments to be separated by size during electrophoresis. The larger fragments move more slowly through the gel because they experience more resistance or “drag”.
=> Constant charge:mass ratio → can separate DNA by size
Electrophoresis is performed after a PCR reaction to visualize the amplified DNA fragments. If the PCR was successful, there should be enough of the target DNA to be observable on an electrophoresis gel. After PCR, there will (hopefully) be enough DNA to see on a gel:
Can you use spectrophotometry?
== it doesn’t provide information about the size of the DNA fragments or whether they are intact DNA strands. All it can do is detect the presence of bases, based on their aromatic rings which absorb UV light at a specific wavelength (260 nm for DNA).
Visualising Results: Staining
Dyes are added to see the DNA or RNA: After the electrophoresis run, the gel is stained with a dye that binds to DNA or RNA, making it visible under certain lighting conditions.
Ethidium bromide intercalates with DNA and fluoresces under UV light: Ethidium bromide, when intercalated or slotted between the base pairs of the DNA, can fluoresce under UV light, allowing the DNA to be seen.
However, ethidium bromide is known to be a powerful mutagen, == Safer alternatives, like HydraGreen and GelRed, have largely replaced it in many labs. These newer dyes also intercalate with DNA and fluoresce under UV light, but are significantly less toxic and mutagenic.
Application: Genotyping
length of variable number tandem repeats will vary between individuals: VNTRs are locations in the DNA where a short sequence of nucleotides is repeated a variable number of times. The number of repeats can be different among individuals, making VNTRs useful for genetic fingerprinting.
Use primers that flank the variable region (i.e., bind to the common region): The length of the PCR product corresponds to the number of repeats because each repeat contributes a certain fixed length to the amplified DNA. (Primer positioned on the start and end of VNTR and VNTR are all same length so if one is 10 meters wide, 2 VNTRs are 20 metres and 3 VNTRs are 30 metres)
the variation in the number of VNTRs is reflected in the length of the amplified DNA.
Size of amplified DNA will increase with the number of repeats: If an individual has more VNTR repeats, the PCR product will be longer, because each repeat adds to the length of the amplified DNA.
Reverse Transcriptase and properties
Reverse transcriptase is a type of DNA polymerase that uses an RNA molecule as a template for making a DNA molecule. This DNA is referred to as cDNA or complementary DNA.
Application :
Retroviruses use this to produce DNA copies of their RNA genome: Retroviruses are a family of viruses that replicate their RNA genomes into DNA using reverse transcriptase. This DNA then gets integrated into the host cell’s genome, enabling the virus to replicate.
Properties:
* Need primers: Like all DNA polymerases, reverse transcriptase requires a primer, which is a short nucleotide sequence that provides a starting point for DNA synthesis.
- Choice of primers depend on application: The specific sequence of the primer you use depends on what you’re trying to accomplish.
- Ability to choose primers to amplify what you want, e.g., mRNA: For example, if you’re interested in copying mRNA molecules, you could use a primer called “oligo(dT)” like a specific key for your lock. This would bind to the polyadenylated tail of mRNA molecules, providing a starting point for reverse transcriptase to make cDNA.
- Random hexamers for all RNA: If you’re interested in copying all RNA molecules, not just mRNA, you could use random hexamers (random strings of 6 nucleotides) as primers. A more general key if you want to copy all types of RNA
Ribonucleases
enzymes that degrade RNA
=> are ubiquitous/omnipresent, found virtually everywhere including on your skin, on laboratory equipment, and in water.
Because ribonucleases break down RNA, any RNA sample that comes into contact with ribonucleases will be degraded. This can be a problem in molecular biology experiments that require intact RNA.
How to prevent RNA degradation:
it’s crucial to avoid contaminating the RNA sample with ribonucleases. One strategy is to convert the RNA to cDNA (complementary DNA), using the enzyme reverse transcriptase. cDNA is more stable and is not degraded by ribonucleases.
Way to check for RNA degradation
Agarose gel electrophoresis can be used not just to size separate DNA, but also to assess the integrity of RNA. For instance, when RNA is isolated from mammalian cells, you should see bands corresponding to 18S and 28S ribosomal RNA (rRNA). If these distinct bands are missing or if you see smearing, it’s a sign that the RNA has been degraded.
RT-PCR
Reverse Transcriptase-Polymerase Chain Reaction
==> commonly used procedure to study the expression of mRNA
two stages of DNA synthesis
1. isolating RNA, then using specific primers to generate complementary DNA (cDNA). The choice of these primers usually depends on the targeted sequence of interest and controls (something that is expressed at a consistent level and unaffected by treatment).
2. PCR amplification of the cDNA, where the gene of interest and controls are amplified and then quantified. During this stage, dyes which bind to double-stranded DNA are used. The fluorescence signal from these dyes increases as the DNA amplifies, allowing for real-time tracking of the PCR process. More abundant sequences are detected earlier in the process due to the greater amount of template DNA, resulting in quicker amplification and earlier detection.
Additionally, it’s important to note the role of dilutions in ensuring the measurements are quantitative. For example, if you run a sample undilified and the same sample at a 1 in 2 dilution, the diluted sample should take one more cycle for detection compared to the undiluted sample. This is due to the doubling of DNA at each cycle, and any inconsistency could indicate some issue in the experimental setup.
how is RT-PCR used
RT-PCR stands for Reverse Transcriptase-Polymerase Chain Reaction, a technique commonly used for testing infections by RNA viruses, such as SARS-CoV2, the virus responsible for COVID-19.
- involves the use of specific primers that bind to a small part of the viral genome. => The primers are designed so that they can identify sequences unique to the virus, such as the envelope (the outside of the virus), RNA-dependent RNA polymerase (the enzyme that replicates the viral RNA), and nucleocapsid (which packages the genome).
- uses controls:
=> positive controls are used to validate the test
=> negative controls (like using water instead of a sample) are used to check for contamination
Similar methods are also used to test for other viruses, such as Ebola.
Detection of new variants, however, often requires development of new specific primers to target the unique sequences of the variant.
Impact of Nucleotide Modification and sanger sequencing
During extension (the process of adding new nucleotides to a growing DNA strand), new nucleotides are added at the 3’ OH (the hydroxyl group at the 3’ end of the DNA strand). Sanger sequencing, a method of DNA sequencing, uses dideoxy analogs.
A dideoxy analog is a modified nucleotide lacking the 3’ OH group. This is significant because when a dideoxy analog is incorporated into a growing DNA strand, it terminates the extension of the strand since the 3’ OH group is required to form the phosphodiester bond with the next nucleotide.
However, Sanger sequencing only uses a small proportion of these dideoxy analogs, allowing the DNA strand to still extend until a dideoxy analog is incorporated at a random point. This random incorporation of dideoxy analogs at various positions in different DNA strands allows for the generation of a ‘ladder’ of fragments, which can then be used to determine the sequence of the original DNA strand.
You assemble from smallest to biggest size, and it forms the sequence of the bases
like puzzle like 1st strand has only 3 and the last was A, 2nd strand has only 5 was G, 3rd strand has only 6 was T, 4th strand has only 4 was A
assemble from biggest so 1st strand, 4th strand, 2nd strand, 3rd strand forms AAGT yay
