Topic 4 Flashcards
What is DNA cloning?
The process in which a desired DNA fragment is selectively amplified and purified to homogeneity from mixed DNA collections.
What are the two major ways of DNA cloning?
Cell-based DNA cloning and PCR
Why is DNA cloning important?
The desired DNA sequence or fragment must be selectively amplified to produce large numbers of identical copies, resulting in purification of the desired product. Therefore, its structure and function can be comprehensively studied.
What is the important enzyme that PCR utilizes?
Taq Polymerase-it is a heat resistant enzyme
What are the requirements for PCR?
template DNA, two primers, dNTPs, and Taq DNA Polymerase.
What are the cycles of PCR?
- Denaturation-Pulls the two DNA strands apart. This occurs around 94°C.
- Annealing-Primer anneals to the target strands. This occurs between 50°C-70°C
- Extension/Elongation- Taq DNA Polymerase elongates the annealed primer by adding dNTPs.
These 3 major steps are repeated 30-40 times. Time of extension depends on size of amplicon.
What are the major advantages of PCR?
It is rapid, sensitive, and robust. You can also adjust the stringency (through annealing temperature).
How do you determine the number of DNA after each cycle?
2^(however many cycles power)
What are the three main PCR based methods that we are focused on?
Allele-specific PCR and Real-time PCR along with the Taq man probe and SYBR Green chemistry with that.
Name a few other PCR methods.
RT-PCR, Inverse PCR, and Differential display-PCR
What is a degenerate primer (Degenerate oligonucleotide-primed PCR)?
If you are unsure if a locus is C or T, design a degenerative primer that is 50% A 50% T so it can bind it it no matter what.
What is touch-down PCR?
First 5 cycles are at 65°C, the next 5-10 cycles the temperature is lowered, and then the next set of cycles are lowered again. This adjusts the stringency of the primer to potentially amplify more target DNA.
What is RACE-PCR?
Rapid Amplification of cDNA End-You sequence the cDNA in the middle part so you can design primers to amplify the 5’ and 3’ end.
What is RT-PCR?
Using reverse transcription to make cDNA and then you can do regular PCR.
What is allele-specific PCR used for?
Allele-specific refers to the specificity for the correct base pairing between the primer and DNA strand at the 3’ end. If the 3’ end is not matching, no extension will occur. This allows the use of PCR to distinguish between alleles of the same gene that differ in a single nucleotide. (ARMS-amplification refractory mutation system)
What is the method for allele-specific PCR?
You have two alleles strands with a single nucleotide polymorphism (SNP). An allele-specific primer is designed for each allele’s complimentary strand so that it can only amplify its respective alleles. You also design a conserved region (regions where alleles are identical) that can bind to allele 1, 2, or both to amplify the conserved region of the alleles. You can use this method to differentiate between allele 1 and 2. You can also tell if you are heterozygous or homozygous for an allele based on if allele 1, allele 2, or both are amplified.
What is Differential-Display PCR?
This uses mRNA to make cDNA to look at expression of different embryonic genes at different stages. This indicates when genes are expressed in development.
Describe inverse PCR.
If you have a piece of genomic DNA and you have a known sequence between “X” and “Y”, but you want to now the upstream and downstream of that sequence, you can digest the sequence with different restriction endonuclease. Then you use ligase to ligate them together and design a primers that go opposite together and run the PCR.
What is a significance of using Real-time PCR?
You have a way to quantify the DNA at the end of each cycle.
Detail the methods of Real-time PCR.
This detects the amount of amplicons accumulating during PCR cycles in “real time”. This technique can be used to quantify the starting amount of DNA, and thus the starting amount of organisms providing that template. That is why it is also called quantitative PCR (Q-PCR). This technology is also utilized in Q-RT-PCR. At the beginning, you have a high amplification efficiency, but as the cycles go on, the enzyme efficiency decreases from denaturation. Fluorescent dye is added for both types so you can see the DNA and quantify the replication in real-time.
What is the chemistry of TaqMan probe?
Made about 25-30bp’s long and designed to the specific middle gene sequence of the amplicon. It is made to have a high-energy dye at the 5’ end (this is known as reporter) and a low-energy molecule at its 3’ end (known as quencher). Taqman probe will anneal to the template strand and at this point, the reporter end and quencher end cancel each other out, yielding no fluorescent color. When Taq Polymerase comes along, it will elongate the new strand until it reaches the probe. Taq Polymerase is very violent and will cut Taq Probe, thus allowing the fluorescent color to show.
What is the chemistry of SYBR Green?
SYBR Green dye has different chemistry than TaqMan. Its fluorescent color shows up when SYBYR Green dye binds to the minor groove in double stranded DNA. This means that SYBR Green dye shows up after elongation, because that is when double stranded DNA happens again. This is used in traditional and real-time PCR.
What are the advantages and disadvantages of SYBR Green?
SYBR Green dye doesn’t require the design of a probe, which saves money. This means you can use it in traditional and real-time PCR. However, using SYBR Green dye also means that it will bind to any DNA, including non-target DNA. This means you need to do a melting curve at the end so you can look at non-specific binding/amplification. You can adjust the melting temperature based on that.
What are the advantages and disadvantages of Taqman Probe?
Taqman probe will only bind to the specific DNA sequences, therefore it won’t amplify non-specific/non-target sequence. This is particularly good for real-time PCR because it won’t quantify non-target DNA. However, this is expensive and requires synthesis of individual probes.
How can you tell which sample has more DNA in it?
Whichever sample reaches threshold first because that means it took less cycles to reach threshold. This is quantified by using real-time PCR. DNA is doubled every cycle (2-fold difference). The difference can be expressed as 2^however many more cycles it takes. (Know how to calculate this according to data).
What are the two major limitations for PCR?
The amplicon can be too big for PCR. An amplicon that is too big would take too long and require special Taq Polymerase (long-range) which is more expensive and more difficult to acquire. The other issue is the low yield of DNA amplification relative to Taq Polymerase.
