Unit 2: The Genome Chapter 6 - Polymerase Chain Reaction - PowerPoint Presentation

1. Unit 2: The GenomeChapter 6 - Polymerase Chain Reaction

2. Figure 6.01. Polymerase Chain Reaction (PCR)During PCR, two primers anneal to complementary sequences at either end of a target sequence on a piece of denatured sample DNA. DNA polymerase synthesizes a complementary strand of DNA from the primers, resulting in two new strands of DNA. In further cycles, the newly made DNA molecules are denatured, primers are annealed, and DNA polymerase copies the target regions, resulting in multiple copies of the original target sequence.

3. Figure 6.02. PCR Machine or Thermocycler(A) The thermocycler or PCR machine can be programed to change temperature rapidly. The heat block typically changes from a high temperature such as 95°C (for denaturation) to 60°C (for primer annealing), then back to approximately 72°C (for DNA elongation) in a matter of seconds. This may be repeated for many cycles.(B) Rows of GeneAmp PCR machines copying human DNA at the Joint Genome Institute, in Walnut Creek, California, which is a collaboration between three of the US Department of Energy’s National Laboratories.(Credit: David Parker, Science Photo Library.)

4. Figure 6.03. Kary Mullis Sees PCR in a VisionGraphical representation of what Kary Mullis described as his first vision of PCR.

5. Figure 6.04. Denaturing the Template and Primer AnnealingIn the first two steps of PCR, a small amount of template DNA is heated to 95°C, which separates the two strands of the double helix. When the temperature is lowered to approximately 60°C, the primers anneal to the ends of the target sequence. Since the primer is present in large excess over the template DNA, the majority of template strands will bind to primers rather than re-annealing to each other. Notice that the PCR primers anneal so they are antiparallel with the target DNA.

6. Figure 6.05. Extension/Elongation by DNA PolymeraseOnce the primers have annealed to the template, the temperature is increased to the optimal temperature for thermostable DNA polymerase, typically around 72°C. The polymerase synthesizes a complementary DNA strand to the template DNA using the pool of dNTPs as building blocks for the new DNA and using the 3′ end of the primer as a starting point. Extension will continue past the end of the target sequence for the first few cycles of PCR. Although the PCR primer is not synthesized, it is incorporated into the final new DNA strand without any modifications.

7. Figure 6.06. Second Cycle of PCRThe two DNA pieces produced in the first cycle are denatured, primers annealed to their complementary sequence in the target DNA, and then DNA polymerase extends the primers to create four double-stranded DNAs. Newly made DNA is shown in blue, and single-stranded DNA from previous cycle is shown in pink.

8. Figure 6.07. Products From the Third Cycle of PCRThe PCR amplicons or PCR products from the second cycle go through the same process as before. The four double-stranded pieces are denatured into eight single-stranded pieces (pink strands). The primers anneal and DNA polymerase makes the complementary strands. This cycle has the first double-stranded copy of the target region without any single-stranded overhangs. Newly made DNA is shown in blue.

9. Figure 6.08. MisprimingIf annealing temperatures are too low, PCR primers can partially anneal to regions outside of the true target DNA and prime DNA synthesis. In this example, The forward primer only partially anneals to the location outside of the target sequence if the annealing temperature is set too low, and will create a longer PCR product than designed.

10. Figure 6.09. Primer-Dimers and Hairpin PCR Primer StructuresWhen sequences within a single PCR primer are complementary, then the primer will fold back on itself to form a hairpin. If the sequence between two PCR primers is complementary, then the primers preferentially bind to each other rather than the target DNA to form a primer-dimer.

11. Figure 6.10. Tailed PCR PrimersPrimers for PCR can be designed to have non-complementary regions at the 5′ end. After PCR, the final PCR amplicon will have the 5′ ends added onto the target DNA sequence.

12. Figure 6.11. Designing Degenerate DNA PrimersDegenerate primers can be designed based on a short amino acid sequence from a protein. Because many amino acids are encoded by several alternative codons, the deduced DNA coding sequence is ambiguous. For example, the amino acid tyrosine is encoded by TAC or TAT. Hence, the third base is ambiguous, and when the primer is synthesized a 50:50 mixture of C and T will be inserted at this position. This ambiguity occurs for all the bases shown in red, resulting in a pool of primers with different, but related sequences. Hopefully, one of these primers will have enough complementary bases to anneal to the target DNA sequence that corresponds to the gene for the protein.

13. Figure 6.12. Inverse PCRInverse PCR allows unknown sequences to be amplified by PCR provided that they are located next to DNA in which the sequence is already known. The DNA is cut with a restriction enzyme that does not cut within the region of known sequence, as shown in Step 1. This generates a fragment of DNA containing the known sequence flanked by two regions of unknown sequence. Since the fragment has two matching sticky ends, it may be easily circularized by DNA ligase. Finally, PCR is performed on the circular fragments of DNA (Step 2). Two primers are used that face outwards from the known DNA sequence. PCR amplification gives multiple copies of one linear product that includes unknown DNA from both left and right sides.

14. Figure 6.13. Reverse Transcriptase PCRRT-PCR is a two-step procedure that involves making a cDNA copy of the mRNA, then using PCR to amplify the cDNA. First, a sample of mRNA is isolated. Reverse transcriptase is used to make a cDNA copy of the mRNA, which is then amplified by a thermostable DNA polymerase in a regular PCR reaction.

15. Figure 6.14. Oligo(dT) Primers and Random Hexamers(A) Entire mRNA samples can be converted into cDNA using oligo(dT) primers that anneal to the polyA tail of mRNAs. The cDNAs will tend to have sequence that is from the 3′ end of mRNAs. (B) Since random hexamers primers have every potential six-base sequence, the final cDNA sample will have complementary sequences from random parts of every mRNA in the sample.

16. Figure 6.15. RT-PCR for Gene ExpressionRT-PCR can determine the amount of mRNA for a particular gene in two different growth conditions. In this example, the gene of interest is expressed in condition 1 but not in condition 2. Therefore, in condition 1, mRNA from the gene of interest is present, reverse transcriptase generates a cDNA, and PCR amplifies this cDNA into many copies. In condition 2, the mRNA is absent and so the RT-PCR procedure does not generate the corresponding DNA.

17. Figure 6.16. Synthesis of Hybrid Gene by Using Overlap PrimersOverlapping primers can be used to link two different gene segments. In this scheme, the overlapping primer has one end with sequences complementary to target sequence 1 and the other half similar to target sequence 2. The PCR reaction will create a product with these two regions linked together.

18. Figure 6.17. Directed MutagenesisOn the left, a mutagenic PCR primer is paired with a reverse primer at the end of the gene. The PCR product has a portion of the gene with the mutation, but this piece must be rejoined to the remaining gene fragment. On the right, the mutagenic primer and reverse primer recognize sequences next to each other in the gene. After PCR, the entire plasmid is amplified from the mutagenic site to the other end of the gene. The gene is recovered by ligating the PCR product.

19. Figure 6.18. Generation of Insertion or Deletion by PCRIn the first step, a specifically targeted cassette is constructed by PCR. This contains both a suitable marker gene and upstream and downstream sequences homologous to the chromosomal gene to be replaced. The engineered cassette is transformed into the host cell and homologous crossing over occurs. Recombinants are selected by the antibiotic resistance carried on the cassette.

20. Figure 6.19. Barcode or Index SequencesBarcodes or index sequences are small segments of DNA with sequences that are unique and not found anyplace else in the host genome.

21. Figure 6.20. Real-Time PCR With SYBR GreenWhen the fluorescent dye SYBR Green I is present during a PCR reaction, it binds to the double-stranded PCR product and emits light at 520 nm. The SYBR Green dye only fluoresces when bound to DNA, therefore, the amount of fluorescence correlates with the amount of PCR product produced. The accumulation of PCR product is followed through many cycles by measuring the amount of fluorescence in each cycle, and plotted on a graph.

22. Figure 6.21. Real-Time PCR With 5′ Nuclease ProbesA 5′ nuclease probe has three elements: a fluorophore at the 5′ end (diamond), a sequence that is complementary to the target DNA (blue), and a quencher at the other end (circle). The fluorophore and quencher are so close that fluorescence is quenched and no light is emitted. This probe is designed to anneal to the center of the target DNA. When Taq polymerase elongates during PCR, its exonuclease activity degrades the probe into single nucleotides. This releases fluorophore from the quencher, allowing the real-time thermocycler to record its signal. The intensity is proportional to the number of new strands synthesized.

23. Figure 6.22. Molecular BeaconA molecular beacon is a probe that has two engineered regions at the ends of the probe sequence. On the 5′ side, a fluorescent tag is added (diamond), and on the 3′ side, a quenching group is added (circle). Just inside the two tags are six base pairs that can form a stem-loop structure. In this state, the probe cannot fluoresce. When the probe binds to the target sequence, the stem-loop structure is lost. Since the quenching group is no longer next to the fluorescent tag, the fluorophore's fluorecent emissions can be detected.

24. Figure 6.23. Forensic Analyses Uses PCRThe two PCR primers will amplify the target DNA from a crime scene or reference sample if the DNA has complementary sequences to the primer. In this example, the first unknown sample did not have complementary DNA sequences, and the PCR primers did not produce any PCR amplicons. In the second unknown sample, the DNA had complementary DNA samples, and therefore, the primers were able to anneal so that DNA polymerase could make copies.

25. Figure 6.24. Mosquito Preserved in AmberThe science fiction film “Jurassic Park” suggested that DNA samples isolated from bugs encased in amber, such as this example, were used to regrow complete dinosaurs.(Credit: Photo by Karen Fiorino.)