Hybridisation DNA sequence

This summary draws upon the provided source material concerning the advanced molecular techniques used to analyse DNA and RNA, focusing on restriction mapping, nucleic acid hybridization, and sequencing technologies (SCIE1106 Lecture 13/L14).

SCIE1106 Lecture 13/14: Analysing DNA and RNA by Hybridisation and Sequencing

The lecture focuses on describing techniques used to analyze and detect specific DNA and RNA sequences, crucial for molecular biology studies.

I. Restriction Enzyme Maps and Genome Analysis

Restriction enzyme digests, combined with agarose gel electrophoresis, can be used to generate a restriction map of DNA genomes,.

  • Mapping Process: This technique works best with small genomes, such as the Lambda bacteriophage (48,502 bp),. The fragments resulting from digestion with individual enzymes ($Eco$R1 or HindIII) and combined double digests ($Eco$R1 + HindIII) are separated by size. By adding fragment sizes and comparing them to those generated by the double digest, researchers can deduce the relative order and pinpoint the exact restriction sites on the genome,,.

  • Challenge of Large Genomes: When large, complex genomes (such as bacterial or eukaryotic DNA) are digested, they create thousands of fragments. These fragments differ only slightly in size and migrate so closely together on the agarose gel that they appear as an unresolved smear,. This smear prevents the identification and isolation of a specific DNA fragment of interest.

  • Solution: To identify a specific fragment within this complex smear, the technique of hybridization must be applied,.

II. Principle of Nucleic Acid Hybridisation

Hybridization is the fundamental technique used to detect specific nucleic acid fragments that cannot be resolved otherwise.

Denaturation and Renaturation

Hybridization relies on the ability of double-stranded nucleic acid molecules to be denatured (separated) and subsequently renatured (re-paired),.

  • Denaturation: Hydrogen bonds and other non-covalent interactions holding the double helix together are disrupted by subjecting the DNA to high temperatures (around $95^\circ\text{C}$) or high pH conditions,. This converts the double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA),.

  • Renaturation: This process is reversible. By slowly cooling the mix of single strands or lowering the pH, the single strands undergo base pairing based on complementarity, reforming double-stranded DNA.

  • Hybrid Formation: Hybridization means forming a double strand from two single strands. This can occur between complementary RNA strands (forming dsRNA), complementary DNA strands (forming dsDNA), or complementary DNA and RNA strands (forming DNA/RNA hybrids).

Probes and Specificity

Detection requires a probe: a single-stranded, labelled nucleic acid (usually DNA), typically 15 to thousands of nucleotides long, which hybridizes to the target sequence,,.

  • Making a Labelled Probe (Random Priming): To label a DNA probe, the target DNA is denatured to ssDNA,. Short, random hexanucleotides (primers) are added, which base-pair randomly with the template,. DNA Polymerase then extends these primers, incorporating labelled dNTPs,. Labels can be radioactive phosphate ($\text{P}^{32}$) or a fluorescent dye attached to the base,.

  • Homologous vs. Heterologous Detection: The outcome of hybridization can be steered by the temperature used during the procedure,:

    • Homologous Detection: Using high temperatures (e.g., $60^\circ\text{C}$ to $65^\circ\text{C}$) ensures the probe only hybridizes to nucleic acid molecules with $100%$ base pairing complementarity (identical sequence),,. This detects identical nucleic acids.

    • Heterologous Detection: Using lower temperatures (e.g., $40^\circ\text{C}$) allows hybridization to occur even when there are slight base sequence differences (mismatches),. This is useful for finding related nucleic acids (e.g., genes belonging to the same family or related genes across different species like mouse and human),,,.

III. Hybridisation Techniques: Blotting and In Situ

Hybridization is implemented through several key techniques, allowing researchers to study DNA, RNA, and their locations in cells.

Nucleic Acid Separation (Agarose Gel Electrophoresis)

All blotting techniques start with separating nucleic acids by size using agarose gel electrophoresis,.

  • DNA Separation: DNA is isolated, digested with restriction enzymes, and separated. It migrates as dsDNA.

  • RNA Separation: RNA is isolated and separated in an agarose gel that contains a denaturing agent,. This agent breaks internal hydrogen bonds and secondary structures within the RNA, ensuring the RNA migrates according to its true size as a single-stranded molecule,,.

  • Concentration: Low agarose concentration (1%) better separates large fragments, while high concentration (2%) better separates small fragments,.

Blotting Techniques (Southern and Northern)

The nucleic acids must be transferred from the fragile gel onto a solid support membrane for subsequent hybridization.

  1. Transfer: The gel is placed on a sponge saturated with buffer. A nitrocellulose or nylon membrane is placed on the gel, followed by a stack of paper towels and a weight,. The buffer is wicked up by the paper towels, carrying the nucleic acids from the gel onto the membrane, where they adhere at the exact positions they occupied in the gel,.

  2. DNA Denaturation (Southern Blot Only): Before transfer for Southern blotting, the dsDNA in the gel must be chemically denatured using an alkaline solution to convert it to ssDNA.

  3. Hybridization and Detection: The membrane is removed and fixed (e.g., with UV light). A labelled, denatured probe is added to a buffer, and the membrane is incubated, allowing the probe to hybridize to its complementary target,,.

  4. Uses:

    • Southern Blotting (DNA): Detects specific DNA fragments,. Used to identify related genes, determine their size(s), and refine restriction maps,.

    • Northern Blotting (RNA): Detects specific RNA molecules (transcripts),. Used to determine the size and number of transcripts, and crucially, to determine patterns of gene expression,. The strength of the signal (band intensity) indicates how highly a gene is expressed (e.g., showing high expression in cancer cells vs. normal cells),.

In Situ Hybridization (ISH)

ISH is a cellular-based technique that localizes DNA or RNA sequences directly within fixed tissues or cells on a slide,,,.

  • Mechanism: Cells or tissue are fixed onto a slide and broken open. The labelled probe is added and hybridized to the DNA or RNA in situ,. Unbound probe is washed away, and results are analysed via microscopy,.

  • RNA-ISH: Localizes mRNA transcripts, answering the question of where a gene is expressed (e.g., pattern formation in a developing fruit fly embryo),. For RNA detection, an anti-sense probe (complementary DNA or RNA) must be used to base pair with the single-stranded messenger RNA.

  • FISH (DNA-ISH): Fluorescent in situ hybridization is used to map specific DNA sequences directly onto chromosomes. Fluorescent probes hybridize to denatured chromosomes spread on a slide. This technique can show the location of genes on maternal and paternal chromosomes, with each gene appearing as two dots per chromatid (representing the two sister chromatids),.

IV. DNA Sequencing Technologies

Sequencing relies on the hybridization of a primer to a single-stranded template, followed by controlled DNA synthesis termination,.

Chain Termination (ddNTPs)

DNA synthesis requires a primer and DNA polymerase, using dNTPs (deoxynucleotide triphosphates) as building blocks. To control the synthesis, dideoxyribonucleotide triphosphates (ddNTPs) are used,.

  • A ddNTP is a modified nucleotide that lacks the 3'-hydroxyl ($\text{OH}$) group on the sugar,.

  • When a ddNTP is incorporated into a growing DNA strand, no further nucleotide can be added, causing chain termination,.

Sanger (Dideoxy) Sequencing

The original Sanger method uses ddNTPs to determine the sequence,,.

  • Original Setup: Four separate reaction tubes are used. Each tube contains the primer, template DNA, DNA polymerase, normal dNTPs, and a small amount of only one specific ddNTP (e.g., ddATP in Tube 1),,.

  • Product: The resulting products are DNA fragments of different lengths, all terminating randomly at the specific base supplied by the ddNTP.

  • Analysis: The four sets of products are separated side-by-side on an acrylamide gel,. By reading the bands from smallest (bottom) to largest (top) across the four lanes, the entire sequence is read (A, T, G, T, C, A, etc.).

Modern Sequencing (Automated Sanger)

Modern sequencing uses fluorescence to simplify and automate the process.

  1. Reaction: All four ddNTPs are included in a single reaction tube, but each ddNTP is labelled with a different fluorescent dye,,.

  2. Separation and Detection: The fragments are separated by size using capillary gel electrophoresis,. A laser excites the fluorescent labels as they pass, and a detector records the colour signal over time,.

  3. Output: A computer generates a chromatogram, displaying the sequence by correlating the colour of the emitted light (the peak) with the base (e.g., red=T, blue=C),.

Shotgun Sequencing

This technique is used to obtain the sequence of small genomes,,.

  • The genome is randomly fragmented into many smaller, overlapping pieces,.

  • These fragments are cloned and sequenced individually (often using Sanger sequencing),.

  • The overall genome sequence is assembled by a computer program that aligns the thousands of sequencing reads based on their overlapping sequences,.