Detailed Notes on CRISPR Cas9, Southern Blot, and Next Generation Sequencing

Overview of CRISPR Cas9

  • Definition: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a gene-editing technology based on a natural system used by bacteria.

  • Components:

    • Cas9 Protein: An enzyme that cuts DNA at a specific location.

    • Guide RNA (gRNA): RNA that directs Cas9 to the specific DNA sequence to edit.

CRISPR Cas9 Mechanism

  • Identify Target Sequence: Scientists identify the problematic part of the human genome causing health issues.

  • Creation of gRNA: A specific gRNA is designed to bind to the identified DNA sequence (comprising As, Ts, Gs, and Cs).

  • Complex Formation: The gRNA is combined with Cas9 to form a complex which is introduced into the target cells.

  • DNA Cutting: The gRNA guides the Cas9 to its target, where it cuts the DNA, allowing for editing (modifying, deleting, or inserting sequences).

  • Functionality: CRISPR Cas9 acts as a "cut and paste" tool, making precise edits to DNA.

Future Applications of CRISPR Cas9

  • Innovative Patient Care: Potential to develop advances that can cure inherited diseases that are lifelong.

Southern Blot Technique

  • History: Introduced by Sir Edwin Southern in 1975.

  • Process Overview:

    1. DNA Isolation: Genomic DNA is isolated and treated with restriction enzymes to create fragments.

    2. Electrophoresis: Fragments separated by agarose gel electrophoresis, resulting in a smear due to numerous fragments.

    3. DNA Denaturation: Alkali treatment separates double-stranded DNA into single strands.

    4. Membrane Transfer: DNA is transferred to a membrane via capillary action.

    5. Heat or UV Treatment: Fixes DNA onto the membrane.

    6. Hybridization: Labeled complementary probes (with radioactivity or fluorescence) are added.

    7. Autoradiography: X-ray film is placed over the membrane to visualize DNA fragments.

Applications of Southern Blot

  • Gene Detection:

    • Presence/Absence: Indicates if a specific gene is present/absent based on autoradiography bands.

    • Copy Number Determination: The number of bands indicates how many copies of a gene exist.

Next-Generation Sequencing (NGS)

  • Revolutionizing Genome Analysis: Capable of sequencing an entire human genome in a day.

  • Process Steps:

    1. Sample Preparation: Isolate DNA and fragment it.

    2. Ligation of Oligonucleotides: Attach sequencing binding sites to fragments.

    3. Flow Cell Application: DNA fragments hybridize to a specialized flow cell.

    4. Amplification: Perform polymerase chain reaction (PCR) to create multiple copies of DNA from fragments (Bridge Amplification).

    5. Sequencing: Use fluorescently labeled nucleotides to determine the order of bases in DNA.

    6. Data Analysis: Millions of reads are compared against a reference genome to construct the full DNA sequence.

Smart Sequencing

  • Smart Cell Technology: Utilizes multiple tiny wells to immobilize single DNA molecules for sequencing.

  • Real-time Measurement: Monitors nucleotide incorporation as polymerase synthesizes new strands, using fluorescence detection.

  • Versatile Sequencing Modes:

    • Circular Consensus Sequencing Mode (HiFi Reads): Produces accurate long reads.

    • Continuous Long Read Sequencing Mode: Generates the longest possible reads.