CR

Polymerase Chain Reaction and Sanger Sequencing

Polymerase Chain Reaction (PCR)

  • Definition: A technique used to amplify a specific segment of DNA, creating millions of copies of a particular sequence.

  • Components:

    • Primers: Short sequences of nucleotides that initiate DNA synthesis.

    • Temperature Steps: Determines the different phases of DNA synthesis.

    • Melting (Denaturation): Occurs at $94^ ext{C}$; DNA double helix unwinds and separates.

    • Annealing: Occurs at approximately $60^ ext{C}$; primers bind to the target sequence of DNA.

    • Extension: Occurs at $72^ ext{C}$; DNA polymerase synthesizes new DNA strands.

  • Thermocycling Process:

    • A series of temperature cycles repeated multiple times to ensure exponential amplification of the target sequence.

    • Cycle Overview:

    • Cycle 1: Results in 2 molecules.

    • Cycle 2: Results in 4 molecules.

    • Cycle 3: Results in 8 molecules. The target sequence is denoted by highlighted organisms in white boxes (matches target sequence).

PCR Template

  • Example Templates:

    • 5' Strand: $5'-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3'$

    • 3' Strand: $3'-GCATTAACACATGGACTCACGGGCATGACCATG-5'$

PCR Extension

  • Extension Mechanism:

    • Example of strand synthesis using templates:

    • $5'-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3'$

    • $3'-GCATTAACACATGGACTCACGGGCATGACCATG-5'$

    • Resulting fragments extending from primers indicate continuous synthesis.

PCR: Exponential Amplification

  • **Amplification Process:

    • Sequence flanked by primers is amplified exponentially.

    • Long templates experience linear amplification.

    • Significant Output Statistics: After 30 cycles yields 1 billion copies, approximately equal to 1 picogram of DNA (assuming an average size of 1000 base pairs).

Sequencing

  • Sequencing Definition: Refers to the method of determining the order of nucleotides in a DNA molecule.

  • Mechanism of Sequencing: Based on the concepts and processes established via PCR. Components include:

    • dNTP (deoxyribonucleotide triphosphate) + template + Taq (polymerase) + primer (only a signle primer used)

    • The PCR process amplifies the target DNA, allowing for sufficient quantity for sequencing analysis.

    • Steps include denaturation, annealing, and extension, which are critical for successful amplification and proper sequencing.

    • Diagrams from slides illustrate these key steps visually for better understanding

    • Denaturation: The double-stranded DNA is heated to around 95°C, causing the strands to separate.

    • Annealing: The temperature is lowered to between 50-65°C, allowing primers to attach to the single-stranded template DNA.

    • Extension: Taq polymerase synthesizes new strands of DNA by incorporating dNTPs, typically at around 72°C.

    • Diagrams:

      • Illustration of the denaturation step depicting the separation of DNA strands.

      • Visual representation of the annealing phase showing primers binding to the template DNA.

      • Diagram highlighting the extension process with Taq polymerase adding nucleotides to the growing DNA strands.

    • .

    • Sanger Sequencing:

    • Measures the length of product DNA strands to determine the nucleotide sequence based on the length of fragments produced.

Sanger Sequencing

  • Basic Concept: A technique that involves the incorporation of di-deoxy ribonucleotides (ddNTPs) that lack a 3' hydroxyl group, resulting in the termination of DNA strand synthesis at random points.

    • The importance of using a low concentration of ddNTPs to achieve termination in varied lengths of fragments.

  • Nucleotide Types in Sanger Sequencing:

    • Ribonucleotide: characterized by a hydroxyl group $OH$ at the 2' position.

    • Deoxy-ribonucleotide: characterized by a lack of hydroxyl group at the 2' position, allowing normal DNA synthesis.

    • Di-deoxy Ribonucleotide: Incorporates two modifications: the lack of $OH$ at both the 2' and 3' positions, preventing further DNA strand elongation.

    • Diagram 1: Structure of Ribonucleotide showing the hydroxyl group at the 2' position.

    • Diagram 2: Structure of Deoxy-ribonucleotide illustrating the absence of the 2' hydroxyl group.

    • Diagram 3: Structure of Di-deoxy Ribonucleotide depicting both the 2' and 3' positions without hydroxyl groups, highlighting its role in terminating DNA synthesis.

    • This unique structure is essential for its function in Sanger sequencing, where the selective incorporation of di-deoxy nucleotides leads to chain termination and enables the determination of DNA sequences.

Sanger Sequencing Steps

  • Melting Step:

    • Example strands used:$

    • 5'-ACATGGACTCACGGGCATGACCATG-3'$

    • $3'-TGTACCTGAGTGCCCGTACTGGTAC-5'$

    • two strands dont have the same sequence

  • Annealing Step:

    • Only a single primer is utilized in Sanger Sequencing.

    • Example primer: $5'-ACATGGACTCACGGGCATGACCATG-3'$

    • Match with the template strand: $3'-GGTAC-5'$

  • Extending Step: Incorporation of labeled ddATP leads to strand termination for produced fragments:

    • Illustrations of Resulting Strands:

      • $5'-ACATGGACTCACGGGCATGACCATG-3'$

      • $ACTGGTAC-5'$

      • Fragments generated of various lengths reflect the sequence of template DNA.

      • Diagram 1: Depicts the amplification process of the target DNA sequence, showing the initial denaturation, annealing of primers, and extension phases.

      • Diagram 2: Illustrates the resulting strands after PCR cycles, highlighting the exponential increase in the quantity of target DNA.

      • ddATP - shows all the strands that end with A

Fragment Analysis via Electrophoresis

  • Process Overview:

    • Electrophoresis is performed to separate DNA fragments by size.

    • Only fragments that are labeled get visualized.

    • Distinct ddNTPs can be distinguished based on their unique fluorescent labels that separate into different lanes:

    • Example nucleotides separated: A, T, C, G.

 In the electrophoresis setup:

  • Each lane corresponds to a specific nucleotide (A, T, C, G).

  • The size of the fragment is indicated by the migration distance on the gel.

  • Diagram 1: Representation of the gel setup showing lanes and DNA migration patterns.

  • Diagram 2: Illustration of different ddNTPs with corresponding fluorescent labels for visualization.

  • first blank part is the primer

  • Dye Labelling Variants:

    • Different dNTPs can be labelled with various fluorescent dyes.

    • Multiple fragments can be analysed simultaneously by running under different colored detection conditions.