Ch. 6 - Polymerase Chain Reaction (PCR) Flashcards
Polymerase Chain Reaction (PCR)
Amplification of a DNA sequence by repeated cycles of strand separation and DNA replication. Usually repeat 25-40 times or so. 30 cycles yield 2^30 copies of the target sequence, exponential amplification. Can yield up to micrograms worth of DNA, which is plenty for further analysis.
Method first described by Kary Mullis in 1983 using the thermostable polymerase. Kjell Kleppe described the method in the 70’s, but without the thermostable polymerase. Per Winge was the first in Norway to use a PCR machine.
Components required:
- DNA fragment: containing the target sequence, the sequence to be amplified, works as a template for the DNA polymerase, often gDNA or cDNA (without introns). Can also amplify RNA molecules.
- Thermostable DNA polymerase: that does not denature at 90 degrees, and has a temperature optimum at around 70 degrees, often use Taq polymerase from Thermus aquaticus.
- Primers: oligonucleotides that are complementary to the ends of the target sequence, and anneal with the DNA at 50-60 degrees. Required for DNA polymerase to bind and to initiate elongation. Primers are specific for the ends of the target gene, and selection of primers is important for efficiency of PCR.
- Free nucleotides: used as building blocks by DNA polymerase in the elongation of the new DNA strand, added to the 3’end.
- A thermo-cycler machine or PCR machine is used for PCR.
3 main steps:
- Denaturation: Temperature is increased to above 90 degrees to separate the two strands in the DNA.
- Annealing/hybridization: Temperature is lowered to 50-60 degrees so the primers can bind to their complementary ends of the DNA template.
- Elongation: DNA polymerase synthesizes DNA complementary to the template strands by adding free nucleotides to the 3’end of the growing DNA strand, starting from the primers.
Primer design and Degenerate primers
- Primers must not hybridize with each other, prevent miss-priming, primer dimers or hair-pin looping
- Forward and reverse primers should have about the same melting point
- Primers should be specific to prevent unspecific annealing
- A couple of GC in the 3´ end is preferred, stronger binding
- If the base sequence is not known, but the amino acid sequence is, degenerate primers can be made
Degenerated primers can be made based on alternative codons for one amino acid. They contain some specific/known bases, and some differ between the primers, often in the third base in the codon triplet.
Degenerated primers is a pool of different primers with alternative bases where they are unknown.
Usage of PCR
- Diagnostics: bacterias and viruses, pathogene microorganisms
- Genetic engineering: cloning (TA cloning or artificial restriction sites), mutagenesis: overlap primers, ligation of DNA fragments, directed mutagenesis by altering bases in PCR primers
- Expression studies: RT-PCR (reverse transcriptase), Real time fluorescent PCR rtqPCR (quantitative)
- Forensics, evolutionary studies, archaeological studies: PCR on region with known polymorphism, sequencing
- “Finger prints” from individuals, can be used to identify individuals, determine paternity or relatedness
- Full genome amplification
- Emusion PCR: Clonal PCR in a liposome, high-throughput sequencing
- Sequencing
- High Resolution Melting point (HRM) analyze, used to identify alleles and mutations, can separate between DNA molecules with only one base in difference, based on melting point for DNA molecules (half the DNA molecules are denatured), whether they are fully complementary or not
Alternative polymerases and PCR modifications
Long (range) PCR: PCR reaction used specifically to amplify longer target sequences than standard PCR.
PCR quick test: antigen/antibody test. Uses one or more primer pairs specific for the organism you are looking for (pathogen). A PCR product with the correct size will identify it.
Hot start PCR: PCR in which Taq polymerase is sequestered (hindered) by antibodies or blocking proteins from the remaining ingredients until the template DNA is fully denatured. Reduces non-specific amplification.
Using alternative thermostable DNA polymerases is useful to increase the fidelity (exactness) of PCR amplification.
TA cloning and artificial restriction sites
TA cloning/repis cloning/T cloning:
- Taq Polymerase generates single 3´-A overhangs with its terminal transferase activity. These can be used for cloning PCR products into TA cloning vectors, a procedure called TA cloning
- TA cloning vector: vector with single 3´-T overhangs (in its linearized form) that is used to clone DNA segments with single 3´-A overhangs generated by Taq Polymerase
- When Taq Polymerase amplifies a piece of DNA during PCR, the terminal transferase activity of Taq adds an extra adenine at the 3´end of the PCR product. The TA cloning vector was designed so that when linearized it has single 5´end thymidine overhangs at each end. The PCR product can be ligated into this vector without the need for special restriction enzyme sites.
Artificial restriction sites are often added to the ends of PCR primers so that the final PCR product can be used directly for cloning into a vector.
Inverse PCR
Method for using PCR to amplify unknown sequences by circulating the template molecule. Performing PCR on a circularized DNA template amplifies neighboring regions of unknown sequence. Can be used to localize elements inserted into a genome in a random way, or if you want to know the genotype based on the phenotype.
Done by cutting the ends with restriction enzymes. The known sequence is in the middle with unknown sequences on both ends. The sticky ends are then joined by DNA ligase, forming a circular DNA template. Primers are added and anneal to the known sequence. DNA polymerases synthesize DNA from the middle of the known sequence, and go each way along the circle. When they meet at the restriction cut sites, the fragments are joined, yielding a linearized fragment with the half of the known sequence on each side, and the unknown sequence in the middle.
Reverse Transcriptase PCR (RT-PCR)
Variant of PCR that allows genes to be amplified and cloned as intron-free DNA copies by starting with mRNA and using reverse transcriptase (cDNA). Thus, identifying the expressed exons of the eukaryotic gene.
The original eukaryotic gene is transcribed in the usual way, and modified so that the introns are removed. The mRNA containing only the exons is then treated with reverse transcriptase to make cDNA. The cDNA is then amplified by PCR. If the gene is expressed, mRNA will be made and cDNA can be amplified, yielding PCR products. If not, no mRNA, cDNA or PCR product will be made.
Reverse transcriptase: Enzyme that makes a DNA copy of the genetic information in an RNA molecule.
complementary DNA (cDNA): Version of a gene that lacks the introns and is made from the corresponding mRNA by using reverse transcriptase.
Differential Display PCR: Variant of RT-PCR that specifically amplifies mRNA from eukaryotic cells using oligo(dT) primers. The oligo(dT) primers anneal to the poly-A tail of the mRNA.
Rapid Amplification of cDNA Ends (RACE): RT-PCR based techniques that generates the complete 5’ or 3’ end of a cDNA sequence starting from a partial sequence.
Real-Time Fluorescent PCR (qRT-PCR)
qRT-PCR: quantitative Real-Time PCR
Amplification method in which the amount of product is measured during the PCR reaction. Fluorescent probes are used to tag the accumulating, synthesized, double-stranded DNA, allowing measurements of the increasing emission from these probes as the amount of DNA copies increases. Can be used to determine the level of expression of different genes, or a gene under different conditions. The amount of fluorescence is proportional to the amount of PCR product.
Two methods to fluorescence the PCR products:
SYBR Green: A DNA-binding fluorescent dye that binds to the double-stranded PCR products and emits light at 520 nm. SYBR Green only fluoresces when bound to DNA, therefore the amount of fluoresce correlates with the amount of PCR product. The accumulation of PCR product i followed through many cycles by measuring the amount of fluorescence. SYBR Green measures the total amount of double-stranded DNA, but cannot distinguish between different sequences. To be sure that the correct target sequence is being amplified, a sequence-specific fluorescent probe is needed.
TaqMan probe: This probe consists of two fluorophores linked by a DNA sequence that will hybridize to the middle of the target DNA after the denaturation step. Fluorescence resonance energy transfer (FRET) transfers the energy from the short-wavelength fluorophore on one end to the long-wavelength fluorophore at the other end. The two fluorophores are so close to each other that the fluorescence is quenched (hindered), and no light is emitted. Fluorescence increases only after the fluorophores are separated by degradation of the linking DNA. When the DNA polymerase meets the probe during DNA synthesis, its nuclease activity cuts the probe into single nucleotides. The probes are then separated, and the short-wavelength fluorophore can now emit light. The fluoresce is proportional to to the number of specific target sequences that has been amplified, and can be detected.
qRT-PCR and gene expression
A mutated phenotype is observed and we want to determine the level of expression of a specific gene that is related to this phenotype e.g. color of plant. A relative quantification can be used to compare the amount of transcripts (mRNA), and the level of gene expression, in mutant and the wild type.
RNA is isolated from the plants (mutant and wild type), and cDNA is made by reverse transcriptase. Real-Time PCR or qPCR is done with gene-specific primers, and the amount of PCR products are measured for both. The crossing point (Cp, where the amount of PCR product crosses the threshold line, in the exponential growth phase) was determined for both, and they were compared. As long as the initial amount of target sequence, and thereby cDNA, was equal for both, and the PCR efficiency was the same, a lower Cp would indicate a higher gene expression because it takes shorter time to reach the threshold.
qRT-PCR and SNP Genotyping
Multiplex PCR: Using different fluorescent dyes on different probes in a qRT-PCR reaction in order to assess the amplification of more than one target sequence.
SNP Genotyping: PCR analysis that can distinguish between alleles with a single nucleotide polymorphism (SNP). “End point genotyping” with two hydrolysis probes.
Differential Display PCR
Variant of RT-PCR that specifically amplifies mRNA from eukaryotic cells using oligo(dT) primers.
