Polymerase Chain Reaction & Human Genome Project

• Polymerase Chain Reaction is the transcription topic.

The Human Genome Project and the History of PCR

• The Human Genome Project started in 1990.
• There were about 30 different institutions involved in the project.
• The goal was to fully sequence the entire human genome.
• It helped scientists understand how diseases develop, and how to prevent and treat them.
• The project led to new technologies and analytical tools.
• An open approach to sharing data was established.
• It helped advance policies and support for sharing scientific data.
• Awareness of ethical issues in biomedical research was raised.
• It helped establish somatic cell genetic disease as a category of genetic disease.
• Mutation was defined as the basis of cancer.
• It helped alter medical practice.
• It inspired other large-scale data acquisition initiatives, including:
  • The International HapMap Project
  • The Cancer Genome Atlas
• The project was initially completed in April 2003.
• The mission was accomplished in May 2021.
• The total size is approximately 3.2 billion bp.
• A lot of sequences are not of interest when detecting specificity.
• The amount of DNA in samples of interest is very small amplification.
• Studying specific sequences is inefficient because of the specificity and amplification issues above.
• Through PCR, studying specific sequences is made possible.
• Dr. Kary Mullis discovered a way to double test target DNA by giving 21 copies which can be repeated N times to get: 2N
• Millions of copies would result after adding an oligonucleotide primer with the 4 dNTPs and DNA polymerase in a chain reaction (N=30 or 40).
• Kary Mullis won the Nobel Prize in Chemistry in 1993 for discovering a way to analyze DNA easily and cheaply.

Steps of PCR

• The image illustrates the polymerase chain reaction (PCR) process, a molecular biology technique used to amplify DNA.
• The process begins with a single DNA strand that serves as the template for amplification.
• In the first cycle, the double-stranded DNA is heated denaturation which separates into single strands.
• Annealing is when Primers bind to specific complementary sequences on the single-stranded DNA.
• DNA polymerase synthesizes new DNA strands using the primers as starting points in a process called extension.
• After the first cycle, 2 copies of the original DNA result, and these serve as templates for another round of PCR in the second cycle.
• The process repeats, leading to 4 copies of the target DNA sequence.
• A third cycle repeats, producing 8 copies, and the DNA strands accumulate exponentially.
• The process continues for around 30 cycles in a standard PCR reaction.
• Amplification occurs demonstrating the power of PCR in DNA amplification since approximately copies of the target DNA sequence are generated by the 30th cycle.

Denaturation Stage

• Denaturation defines the unfolding or breaking up of a protein, modifying its standard three-dimensional structure.
• Proteins may be denatured by chemical action, heat, or agitation causing a protein to unfold or its polypeptide chains to become disordered typically leaving the molecules non-functional.
• In denaturation, Double-stranded DNA is therefore separated into 2 single strands at a temperature of 94-96oC in several seconds to minutes.
• Larger templates result in a longer time.

Annealing Stage

• Hybridization of primers requires complementary DNA sequences and is the most critical step for specificity.
• Temperature of 50-70oC is required.
• The starting point is determined using the Tm of the primer sequences.
• The melting temperature (Tm) controls the temperature at which primers attach to DNA polymerases.
• The melting temperature (Tm) in polymerase chain reaction (PCR) is used to control the temperature at which primers attach to DNA and is a critical parameter in PCR and other molecular biology experiments.
• The reaction temperature needs to be low enough for primers to bind to the DNA template, but high enough to prevent the formation of undesired duplexes.
• All primers in a reaction should have similar Tm values so they can anneal and dissociate from DNA sequences at similar temperatures.
• Amplification proceeds at the selected temperature due to all primers having similar Tm values.
• Melting analysis is done during sequence verification following PCR.

Primers and Extension Stage

• Primers with Tm in the temperature range of 52-58°C generally produce the best results, while primers with Tm above 65°C have a tendency for secondary annealing.
• The 5’-’3 is the leading strand, and must be distinguished from 3’-5’ lagging strand during extension.
• The leading strand is a DNA strand that is synthesized continuously in the 5' to 3' direction
• The lagging strand is synthesized in small fragments called Okazaki fragments.
• The extension stage is performed by DNA polymerase at a Temperature of 68-72oC.
• The polymerase synthesizes a copy of the template DNA, and catalyzes formation of the phosphodiester bonds between dNTPs and the 3’ end of the primer which results in a 2x template number.
• Taq polymerase belongs to the amplification stage of a PCR reaction, synthesizing new DNA strands by adding nucleotides to the template during the extension phase.
• Taq polymerase can function at higher temperatures without denaturing, making it crucial for PCR efficiency.

Components of PCR

• Primers are single-stranded DNA fragments with a 20-30 bp length that determine the specificity of PCR and contain sequences complementary to sites flanking the region of interest such that they work like the primers in vivo.
• The forward Primer Hybridizes with the minus strand at to the sequences amplified with 5’, while the reverse primer hybridizes with the plus strand amplified from with 3’.
• Forward creates the same copy of the target gene-the plus strand sequence with the conditions for binding to its target being, and the reverse primer has similar Melting temp (Tm).
• Routine clinical analyses require at least DNA Template of 100 ng – 1 μg and needs to be in good condition, free of contaminants, no nicks and breaks
• The DExOXYRIBONUCLEOTIDE BASES are building blocks of DNA needed in requirements of 0.1-0.5 mM of each i.e. dATP (adenine), dTTP (thymine), dGTP (guanine), and dCTP (cytosine).

Polymerases

• The First polymerase from E. coli that Needs to be added after each round of denaturation must be distinguished from Taq polymerase
• Taq polymerase must have these characteristics: Thermus aquaticus, Thermostable and Good fidelity through accurate copying of the template.
• Taq polymerase belongs to the amplification stage of a PCR reaction responsible for synthesizing new DNA strands by adding nucleotides to the template during the extension phase.
• If the Polymerase is Taq , it is a heat-stable DNA polymerase enzyme extracted from the bacteria "Thermus aquaticus", which is commonly used in the Polymerase Chain Reaction (PCR) technique to amplify specific DNA sequences by repeatedly copying them.
• In contrast to Taq, Tth polymerase are Thermus thermophilus Also and have reverse transcriptase activity where the Enzyme is Used in RT-PCR (RNA template).

Buffers

• PCR Buffers activity needs to provide optimal conditions provide cations from pH buffers and salts.
• The PCR buffer is a solution of salts, stabilizers, and enhancers that helps maintain the pH of a PCR reaction.
• All that makes help Maintain pH: The buffer neutralizes acidic or basic compounds in the sample.
• The accessory in the buffer includes: Function, Enhancers, and Stabilizers such as: which include bovine serum albumin, dithiothreitol , and tri-ton.
• Monovalent and Divalent Cations affects denaturing and annealing temperatures i.e. salt increase resulting in longer DNA sequences denaturing slower.

Synthesizing ODNs

• ODNs are made in a laboratory using solid-phase chemical synthesis, with the four nucleic acids: A, T, C, and G.
• Nucleic acids are added one by one to form a chain of nucleotides.

Thermal Cycling

• Multiple water baths or heat blocks of the first PC are done manually to control the temperature during the reaction.
• With Thermal cyclers rapid temperature automatization is achieved at each step by maintaining the required amount for designated periods.
• By precisely controlling temperature changes PCR stages like, DNA denaturation, primer annealing, and extension are precisely achieved to amplifying desired sequences.
• For its components A thermal cycler has a heating block with holes where PCR tubes containing the reaction mixture are precisely placed.
• Applications include, DNA sequencing, genotyping, cloning, mutagenesis, and other molecular biology techniques.

PCR Controls and Contaminants

• For PCR with negative and reagent controls, there are problems resulting from contamination.
• Contamination includes template amplification and contamination balance with reagents used in past reactions.
• To resolves contamination and carry out Physical decontamination, induce damage to DNA with UV light to prevent the formation of double strands.
• To resolves contamination and carry out Chemical decontamination , use dUTP-UNG system.

Miscellaneous Methods

• Misprimes from Aberrant binding of the primer carries the carries the primer sequence and becomes a target for the next amplifications
• Primer Dimers form as Artifact in PCR where there is Primers binding to each other giving PCR products double the size of primers.
• Hot Start PCR requires Type 1 reaction mixes.

Complex PCR e.g. Nested PCR

• Nested PCR has two pairs of primers used to amplify a single target in separate PCR programs
• Advantages of Nested PCRinclude: Increased sensitivity & specificity
• The Disadvantage of Nested PCR requires increased duration to run. requires increased duration to run.

Reversed and Real Time PCR

• Reversed Transcriptase PCR requires type 3 sequestered enzymes that are Supplied in their inactive form.
• Real time PCR with high resolution with less cross reactivity produces high quality results.