DNA Synthesis and PCR Techniques Vocabulary

DNA Synthesis and Analysis

Requirements for DNA Synthesis

• Template: A DNA molecule to start with.

• Enzyme: DNA polymerase.

• Primer: Provides a free 3' hydroxyl group for the polymerase to bind and initiate synthesis.

• Nucleotides: Building blocks for synthesizing the new DNA strand.

• Buffer solution: Provides a suitable chemical environment for the reaction.

• Precise temperature control: Important for optimal enzyme activity.

Polymerase Chain Reaction (PCR)

• A cyclic process that mimics DNA replication to produce millions of copies of a DNA sequence.

• Developed by Kary Mullis in 1983; he won the Nobel Prize in 1993.

• Refined by the discovery of Taq polymerase, a heat-stable enzyme from Thermus aquaticus.

• Mullis was a controversial figure, questioning the link between HIV and AIDS and expressing skepticism about human-caused climate change.

PCR Steps

1. Denaturation: High temperature separates the DNA double strand. Analogous to helicase in natural replication.

2. Annealing: Primers bind to the template strands at a lower temperature (forward and reverse primers).

3. Extension/Elongation: DNA polymerase synthesizes new strands at an increased temperature.

Amplification

• The amount of DNA doubles with each cycle, leading to exponential amplification.

  • Cycle 1: 2 copies

  • Cycle 2: 4 copies

  • Cycle 3: 8 copies

  • Cycle 4: 16 copies

Variations of PCR

Reverse Transcription PCR (RT-PCR)

• Template is cDNA (complementary DNA), which is reverse transcribed from RNA (typically mRNA).

• Important for gene expression studies and cloning genes.

• cDNA contains only the exons of a gene.

RT-qPCR (Real-Time Quantitative PCR)

• Measures the number of copies synthesized in real-time.

• Template is labeled with DNA-binding dyes or probes to measure fluorescence.

  • The probe contains a quencher and a fluorophore.

• As the polymerase synthesizes the new strand, the fluorophore is released, and a fluorescence signal is detected.

• The signal is quantified as an RFU (relative fluorescence unit) value.

• The more copies of the target sequence, the stronger the signal.

• A dilution series is used to create a standard curve. The earlier the RFU curve appears, the higher the concentration of the target.

• The PCR efficiency decreases as the reaction runs due to depletion of nucleotides and enzyme fatigue.

Applications of PCR

• Diagnosis of pathogens (bacteria, viruses, fungi).

• DNA fingerprinting for individual identification.

Primer Design and Specificity

• Primer specificity is crucial for polymerase binding.

• Allows differentiation between alleles (variations of a gene).

• Sensitivity to primer design and contamination are important considerations.

Primer Length and Combinations

• Shorter Primer (e.g., 3 bases):
  4^3 = 64 possible combinations are found throughout a genome, likelihood to randomly encounter its complement is high.

• Longer Primer (e.g., 25 bases):
  More possible combinations, likelihood of random binding decreases.

• Longer primers generally increase specificity.

Applications

• Pathogen Diagnosis: Identifying specific DNA or RNA sequences of pathogens. Important to note that RNA requires extra caution due to sensitivity to degradation.

• DNA Fingerprinting: Identifying individuals based on unique DNA sequence patterns, especially short tandem repeats (STRs).

  • Used in forensic science, paternity testing, and personal identification.

  • STRs are highly variable due to their repetitive nature and location in non-coding regions, leading to a high mutation rate and individual uniqueness.

  • DNA patterns can be visualized on agarose gels as fragments of varying lengths.

DNA Fingerprinting and STRs

• Each individual has a unique combination of STRs.

• The Y chromosome can be used to distinguish between males and females.

Case Study: The Green River Killer

• Gary Ridgway was identified using STR analysis of semen fluid collected from victims in the 1980s.

• The DNA matched a saliva swab from Ridgway.

DNA Databases

• CODIS (Combined DNA Index System) is a national DNA database in the U.S. used by law enforcement.

• It stores DNA profiles from convicted offenders and crime scene evidence.

• The system relies on STR markers.

• Initially used 13 STRs, later updated to 20 loci for improved accuracy.

Paternity Testing

• Based on multiple STRs.

• Individuals inherit one allele from each parent at each STR locus.

• A paternity index is calculated based on the probability of the alleged father sharing genetic markers with the child, compared to a random, unrelated individual.

• A combined paternity index (CPI) across multiple loci is used to calculate the probability of paternity.

• A probability of 99.99% is typically required for legal paternity confirmation.

• Uses the equation: Probability \, of \, Paternity = \frac{CPI}{CPI + 1}

Controversial Incident: HIV Transmission by a Dentist

• From 1990-1992, six patients were found to be HIV positive without typical risk factors.

• The only linkage was the dentist, who was diagnosed with AIDS in 1986 but continued practicing.

• The CDC investigated the case using HIV genotyping.

• HIV genetic sequences from the patients and the dentist were isolated, amplified, sequenced, and compared. Local controls were also analyzed.

Phylogenetic Tree

• The sequences are arranged into clusters based on similarities.

• Sequences of HIV isolates from the patients clustered with the dentist's sequence, suggesting they were infected by the dentist.

• The exact mechanism of transmission could not be determined (blood-contaminated instruments or needle-stick injuries).

• It remains an exceptionally rare event in medical history.

DNA Sequencing

• Determining the exact order of nucleotides in a DNA molecule.

• Fundamental technique in molecular biology, genetics, and biotechnology.

• Used for studying genomes, identifying mutations, and diagnosing diseases.

Sanger Sequencing

• Developed by Frederick Sanger in 1977, also known as chain termination sequencing.

• Mimics DNA replication but uses dideoxynucleotide triphosphates (ddNTPs).

• ddNTPs lack a 3' hydroxyl group, terminating the DNA chain.

Ingredients for Sanger Sequencing

• dNTPs (regular nucleotides)

• DNA polymerase

• Primer

• ddNTPs (labeled with fluorescent dyes)

Process

1. ddNTPs are incorporated into the growing DNA chain, terminating it

2. This generates a collection of DNA fragments ending at every possible position where a ddNTP could have been added.

3. The fragments are separated by capillary electrophoresis.

4. A laser detects fluorescent dyes corresponding to the base terminating the fragment.

Key Difference: dNTPs vs. ddNTPs

• dNTPs have a free 3' hydroxyl group, allowing phosphodiester linkages to continue the chain.

• ddNTPs lack the 3' hydroxyl group, preventing further extension of the DNA strand and terminating the chain.

Characteristics of Sanger Sequencing

• High quality (accurate base calling).

• Low output (sequences a single template in each reaction).

• Time-consuming.

• Sequence length ranges from 800 to 1,200 nucleotides.

• Suitable for sequencing single genes but not whole genomes.

Limitations

• Cannot be used for whole genomes due to the time-consuming process.

Next-Generation Sequencing (NGS)

• Introduced around 2005.

• Allows millions of sequencing reactions to be performed simultaneously (massive parallel sequencing).

• Reads are much shorter than Sanger sequencing reads.

• No need for constructing genomic libraries by conventional cloning.

• Sequencing occurs in real time and continuously.

Read Length

• A key parameter in sequencing.

• Generally, the longer the read, the lower the quality.

Third-Generation Sequencing

• Can sequence DNA templates that are kilobases in length.

• However, the quality is not as good as for short reads.

Genome Complexity

• Wheat has a more complex genome than the human genome.

• The human genome has approximately 3.2 billion base pairs.

• The wheat genome has approximately 17 billion base pairs.

• Human genome: 46 chromosomes (23 pairs), diploid (two sets of chromosomes).

• Wheat genome: 42 chromosomes (six sets of seven chromosomes), hexapolyploid (more than two complete sets of chromosomes).

Genome Characteristics

• Estimated gene number in the human genome: 20,000 - 25,000

• Estimated gene number in the wheat genome: 80,000 - 100,000

• Amount of repetitive DNA in the human genome: 50%

• Amount of repetitive DNA in the wheat genome: 80%

• Polyploidy in wheat introduces more homologous regions and redundancy, creating a more complex genomic architecture.

Human Genome Project

• Launched in the early 1990s.

• A landmark research project involving international collaboration.

• The first draft was presented in February 2001.

• Aimed to unravel all human genes, determine their location on the chromosomes, and understand genetic variation.

Genome Sequencing

• Advances have significantly decreased the cost over the years.

• Although having the ability to sequence the DNA of organisms, the full genome is difficult to obtain

Challenges

• Sequence reads are short in relation to a complete genome.

• Millions and billions of sequencing reads need to be oriented and located within the genome.

• Repetitive sequences make it difficult to determine the correct position.

• Putting the pieces together in the right order without knowing how the genome is supposed to be.

Assembly Process

• The process of putting DNA sequences together in the correct order.

• Repetitive or homologous regions in the genome complicate this process, particularly with short reads.