Topic 6-2 PCR, DNA sequencing and libraries

BIOL 207: Molecular Genetics and Heredity

Topic 6: Techniques in Molecular Genetics

Modern Molecular Genetic Techniques

• Locate a gene/DNA sequence

• Remove/Copy DNA sequence

• Visualize DNA

• Store new DNA sequences

• Edit any genome

• Recombinant DNA

• Polymerase Chain Reaction (PCR)

• DNA sequencing and DNA libraries

• CRISPR/Cas genome editing

• Capabilities and Major Advancements

Polymerase Chain Reaction (PCR)

• Development: First developed in 1983 by Kary Mullis, awarded Nobel Prize in Chemistry in 1993.

• Impact: Led to a genetic technological revolution. Today: PCR is fast, cheap, easy. Locates and significantly multiplies specific DNA sequences.

Process Overview

• Inspired by cellular DNA replication

PCR Components

1. Single-stranded DNA template

   • DNA unwound by helicases.

2. Replication machinery

   • Enzyme: DNA polymerase.

3. RNA primers

   • Synthesized by primase, short chains (17-25 nucleotides) complementary to the DNA template.

4. Free dNTPs

   • Nucleotide triphosphates needed for synthesis.

PCR Reaction Mixture

• Key Components:

  1. DNA template (double stranded)

  2. Primers (forward and reverse)

  3. dNTPs

  4. Heat-resistant DNA polymerase

  5. Buffer (ions)

• Volume: PCR tube generally contains 20-50 µL.

Steps of PCR

1. Separation of DNA: Heat to 90-100°C for a few minutes.

2. Annealing of Primers: Cool to 30-60°C for primer binding.

3. DNA Synthesis: Raise to 72°C for polymerase activity.

• Polymerase synthesizes new DNA strands from the template.

  • Key Note: PCR relies on thermal-resistant DNA polymerase found in Thermus aquaticus from Yellowstone.

Cycling Process

• Thermocycler allows repetition of the three steps (separation, annealing, synthesis) 25-35 times.

• Result: Robust amplification, yielding approximately 2^n copies (n = number of cycles).

Specificity of PCR

• Amplification is specific due to the use of forward and reverse primers (each ~20 nt long).

Creating DNA Libraries

• Purpose: To discover new genes.

• Method:

  1. Break up the genome into small pieces.

  2. Clone them into plasmids – „Shotgun cloning“ creates a comprehensive library of clones containing DNA sequences from the source genome.

Process of Creating DNA Libraries

1. Isolate genomic DNA from organism/tissue.

2. Fragmentation: DNA cut into overlapping pieces via restriction enzyme digestion.

3. Cloning: Insert fragments into vectors (plasmids).

4. Transformation: Introduce to bacteria, store at -80°C in glycerol.

5. Screening: Identify colonies with the desired gene using probes.

cDNA Libraries

• Static Nature of Genomic Libraries: Same DNA sequences no matter the source.

• cDNA Libraries Creation:

1. Isolate mRNA from specific cells.

2. Reverse transcription to convert mRNA into cDNA using oligo-dT primers and reverse transcriptase.

3. Clone cDNA into vectors and transform into host cells.

DNA Sequencing Techniques

• Sanger Sequencing:

• Developed by Frederick Sanger in the 1970s.

• Uses dideoxyribonucleoside triphosphates (ddNTPs) that terminate synthesis when incorporated.

• Fragments of DNA are produced corresponding to the length of the template.

Modern Automated Sanger Sequencing

• Utilizes fluorescently labeled ddNTPs to allow for simultaneous analysis of all bases in one reaction mixture.

• Fragments pass through a detector for sequence reading.

Importance of DNA Sequencing

• Provides the foundation for genetic analysis, gene identification, and understanding genome structure and function.