Molecular Biology: Nucleic Acid Hybridization and Protein Analysis

Nucleic Acid Hybridization

  • A principle underlying many important techniques in molecular biology.
  • Takes advantage of the complementary nature of DNA and RNA.

Principles of Hybridization

  • Hybridization depends on base pairing.
  • A denatured (single-stranded) probe is added to a complex mixture of (denatured) target nucleic acid sequences (DNA or RNA).
  • Denaturation is usually effected by controlled heating.
  • The mix is incubated under conditions that promote the formation of hydrogen bonds between complementary strands (also known as annealing).

Denaturation and Tm

  • Denaturation of DNA leads to an increase in optical density.
  • OD260OD_{260} is often used to measure optical density.
  • TmT_m refers to the melting temperature.

Hybridization Process

  1. Two strands of a DNA molecule denature (separate) upon being slowly heated.
  2. A probe with a complementary base sequence is added to the denatured DNA.
  3. The probe binds to the target DNA strand, forming a double-stranded hybrid molecule.

Factors Affecting Hybridization

  • Strand Length: The longer the probe, the more stable the duplex.
  • Base Composition: GC base pairs are more stable than AT pairs. A higher %GC leads to greater stability.
  • Chemical Environment:
    • The concentration of salt (e.g., sodium chloride, buffers containing Na+Na^+).
    • Chemical denaturants (formamide or urea) destabilize hydrogen bonds and can be used to control annealing conditions.

Steps Involved in Hybridization

  1. Incubate denatured probe with target under controlled buffer, salt, and temperature conditions.
    • Low stringency to ensure substantial hybridization.
  2. Wash at higher stringency to eliminate non-specific and badly mis-matched hybrids.
  3. Process (the membrane) to visualize any bound probe, visualizing signals from labels.

Identification of Specific Sequences

  • Conventional DNA probe.
  • Oligonucleotide probe: Using oligonucleotide probes requires sequence information.
    • Perfect match: Stable.
    • Single mismatch (e.g., allelic): Stable at reduced hybridization stringency and unstable at high hybridization stringency.
    • 20% mismatch (e.g., coding sequences of human and mouse genes): Stable at reduced hybridization stringency.

Stringency

  • Probe – target annealing, stability of hybrids.
  • Stringency of the incubation/washing conditions.
  • Higher temperature = more stringent.
  • Lower salt concentration = more stringent.
  • Higher stringency - higher level of base complementarity is required for duplexes to form and remain stable.
  • Lower stringency permits a higher degree of mis-match: less similar/less related sequences are able to form stable hybrids.

Factors Influencing Hybridization

  • Heat:
    • Increases movement of molecules.
    • Destabilizes hydrogen bonds between base pairs.
  • Salt:
    • High salt decreases repulsion between phosphate backbones.

Visualization of Probes

  • RNA and DNA probes can be directly labeled by incorporation of a chemically labeled base.
  • Synthesis of new RNA or DNA containing the fluorescently labeled base.
  • End-labeling by swapping the terminal phosphate group with a radioactive phosphate à autoradiography.

Digoxigenin

  • A plant steroid for which very specific antibodies are available.
  • Incorporated into probes as DIG-labelled UTP.
  • Detected with antibody conjugated to alkaline phosphatase.
  • Indirectly labelled and visualized.

Detection of Indirect Labels

  • Horse Radish Peroxidase (HRP):
    • DAB – diaminobenzedine (substrate).
    • Brown liquid – brown precipitate.
    • Luminol-based chemiluminescent substrates.
  • Alkaline Phosphatase:
    • NBT/BCIP (substrate).
    • nitro-blue tetrazolium chloride/ 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt.
    • Yellow liquid – dark purple precipitate.

Techniques Utilizing Nucleic Acid Hybridization

  • DNA/Colony hybridization.
  • Southern Blotting - developed by Ed Southern
    • DNA fragments separated on an agarose gel are transferred to a filter and hybridised to a DNA probe via Watson-Crick base pairing.
    • Probe is labelled to allow detection.
  • Northern blotting
    • RNA species (transcripts and/or mRNAs) on filter.
    • DNA probe.
  • In situ hybridisation.

Colony Hybridization

  • Begin with a Genomic Library or cDNA library: “A collection of living bacterial colonies that have been transformed with different pieces of DNA from the organism of interest”.
  • Screening is used to find the colony with the gene of interest amongst thousands of colonies.

Screening Clones from a Library by Colony Hybridization

  1. Replicate a plate of colonies from library onto nitrocellulose/nylon filter/membrane and incubate.
  2. Treat filter containing colonies to release DNA, denature DNA, fix single-stranded DNA on filter by baking.
  3. Incubate filter in solution containing labelled, denatured, DNA probe.
  4. Wash filter, dry, and detect.
  5. Locate relevant colony on master plate.

Colony Hybridization Process

  • Petri dish with colonies of bacteria containing recombinant plasmids.
  • Peel paper from dish to produce replica of colonies.
  • Lyse bacteria and denature DNA.
  • Incubate with radioactively labeled DNA probe and wash.
  • Expose paper to photographic film to detect position of desired colonies by autoradiography.

cDNA vs. Genomic DNA

  • cDNA:
    • Synthetic DNA whose base sequences are complementary to mRNA.
    • Represents the transcriptome.
    • Synthesized from the reverse transcription of different types of RNA.
    • Consists of exons or coding regions.
    • Contains a few base pairs.
    • Used to produce cDNA libraries.
  • Genomic DNA:
    • Total set of chromosomal DNA in the genome.
    • Represents the genome.
    • Can be extracted from existing genomes.
    • Consists of coding and non-coding sequences of an organism.
    • Contains a large number of base pairs.
    • Can be used to produce genomic libraries.

Screening

  • Genomic clones are not expressed by bacteria.
  • Nucleic acid hybridization is used with a probe to detect it.
  • cDNA clones can be expressed using an expression vector, promoter and terminator signal.
  • Proteins detected by antibodies or assays (enzymes).

Detecting Protein Produced from a cDNA Clone

  • For a cDNA clone in an expression vector one can detect protein produced.
  • Transform E.coli – plate cells.
  • Pick some cells from a colony and grow on membrane.
  • Lyse cells.
  • Proteins bind to membrane.
  • Detection with antibody using a primary antibody and a secondary antibody.
  • Detect the secondary antibody.

Electrophoresis

  • DNA is negatively charged due to phosphates on its surface.
  • As a result, it moves towards the positive pole.

Gel Electrophoresis

  • Lightest fragments migrate furthest.
  • Heaviest fragments migrate the least.
  • Direction of movement is from negative to positive.

The Gel

  • Agarose: A polysaccharide from agar, which comes from red seaweed.
    • Inert matrix useful for separation techniques.
  • Buffer: TAE or TBE
    • Tris acetate EDTA
      • Size separation, check Maniatis
      • Disadvantages: More prone to overheating (Max 5-10v/cm, 100v for gel 10cm in length)
    • Tris borate EDTA
      • Size separation check Maniatis
      • Disadvantages: Borate is an enzyme inhibitor

A New Generation of Buffers

  • 10mM Sodium borate:
    • 100bp - 5kb
    • 35v/cm
  • 5mM Lithium acetate
    • >3kb
    • 30v/cm (Brody and Kern 2004)

Southern and Northern Blotting

  • Probes are typically:
    • Cloned genomic gene sequences.
    • or cloned cDNAs (i.e. complementary to a specific mRNA).
  • Southern blotting experiments with cDNAs led to the discovery of split genes (experiments of Jeffreys, and Flavell).

Southern Blotting and Hybridization: The Experimental Procedure

  1. Digest genomic DNA with Restriction Enzyme (cut ALL the sites present in the DNA).
    • Important for all sites to be cut so fragments always have the same relationship to genes.
  2. Separate the “restriction” fragments according to size, by electrophoresis on an agarose gel.
    • Many fragments of all sizes create a smear
    • Ethidium bromide (or other intercalating dyes eg GelRed, SYBR green or SYBR safe) is used to make DNA fluoresce.
  3. Transfer the DNA fragments from the gel to a nitrocellulose or nylon membrane by capillary flow – ‘Southern blotting’.
    • The gel is initially soaked in acid, then alkali, to break the fragments down to a size suitable for transfer to the membrane.
    • The alkali treatment also denatures the DNA so that single-stranded DNA is available for base-pairing with labelled “probe”.
    • The gel is then positioned in the transfer apparatus.
    • Flow of solvent via the wicks, up through the gel into the paper towels, results in point-for-point transfer of the DNA to the membrane.
    • DNA sticks to the membrane and does not pass through.
  4. Dry and bake the filter in an oven – this fixes the DNA firmly to the nitrocellulose membranes.
  5. The filter can now be hybridized to labelled (denatured) probe and processed to visualise chemical labels such as digoxigenin or biotin.
  6. Excess probe is washed away and stringency is increased to make sure probe is only bound to the matching DNA.
  7. Detection of the probe eg with chemiluminescent substrate for DIG labelled probe and capture of image with photographic film or electronic image capturing.

Southern Blotting Process

  • Solution passes through gel and membrane to paper towels.
  • DNA is transferred to the membrane.
  • Hybridize with unique nucleic acid probe.
  • Remove unbound probe.
  • Expose to photographic film.

Application of Southern Blotting

  • Detecting polymorphisms in DNA.
  • Detecting presence or absence of DNA sequence eg looking for transgene in possible transgenics.

Polymorphism

  • Existence of two or more variants.
  • Alleles.
  • Phenotypes.
  • Sequence variants eg Single nucleotide polymorphisms (SNP).
  • Can be silent or disease causing.
  • If they disrupt a RE site à restriction fragment length polymorphism (RFLP).

Restriction Fragment Length Polymorphism (RFLP)

  • Illustrates how variations in DNA sequence can lead to different fragment sizes after restriction enzyme digestion.
  • Different alleles (A and a) may have different restriction enzyme cleavage sites, resulting in larger or smaller DNA fragments.
  • This leads to different band sizes on a gel after Southern blotting.

RFLP cont.

  • Homozygous AA, Heterozygous Aa, Homozygous aa.
  • DNA bands from genotype AA, DNA bands from genotype Aa, DNA band from genotype aa.

DNA Fingerprint

  • Many RFLPs together can create a unique pattern.
  • This is an example of an old-fashioned DNA fingerprint.

Applications of Northern Blotting

  • RNA, not DNA, is the target.
  • To determine if a gene is expressed or not:
    • In a particular tissue
    • At a particular time
    • In certain conditions
  • Useful for revealing size or sizes of RNA – isoforms.

Northern blot example

  • Hind et al (1993) Nature genetics, shows the expression patterns of a gene in different tissues like heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and testis.

Alternatives to Northern Blotting

  • Northern blotting is an older technique but is still used.
  • However, you can detect mRNA levels using qRT-PCR.

In Situ Hybridization

  • Chromosome in situ hybridization: Metaphase or protometaphase chromosomes are probed with labelled DNA. The DNA can be labeled with a fluorochrome (FISH).
  • Tissue in situ hybridization: Sliced or whole mounted preparations can be probed with RNA probes to detect mRNA expression.

In Situ Hybridization Method

  • PCR to amplify the piece of DNA to be used as a probe.
  • Add a T7 RNA polymerase consensus site to the reverse primer.
  • Use T7 RNA polymerase to make an RNA probe.
  • DIG – U.
  • Test probe on a dot blot to check activity, concentration and DIG-U incorporation.
  • Hybridise to tissue.
  • Wash extensively.
  • Detect with NBT/BCIP.

Tissue In Situ Hybridization

  • Example: ß-myosin in 13d embryonic mouse (Heart ventricles).

FISH - Spectral Karyotyping

  • A method to visualize all the chromosomes at once, each in a different color.

Western Blotting

  • Used to detect specific protein molecules from among a mixture of proteins.

Key Steps in Western Blotting

  1. Prepare the protein from your sample.
  2. Run an SDS-PAGE gel (Note: PAGE= Polyacrylamide gel electrophoresis).
  3. Transfer the proteins to the membrane.
  4. Detect the presence of the proteins using antibodies.

Extracting Protein from Cells/Tissues

  • Methods include:
    • Liquid nitrogen grinding.
    • Sonication.
    • Bead beaters.
  • Use Buffer such as: RIPA – salts and detergent.

SDS-PAGE

  • Sodium dodecyl sulfate poly-acrylamide gel electrophoresis.
  • Proteins run as polypeptides.
  • Vertical gel.
  • Current separates proteins by size.
  • SDS coats proteins conferring negative charge.

Antibodies

  • Primary antibody:
    • Raised against protein of interest.
    • Common species used to make primary antibodies: Rat, mouse, rabbit, guinea pig.
  • Secondary antibody:
    • Raised against antibody from primary species.
    • Common species used for secondary antibodies: Chicken, Donkey, Goat, Guinea Pig, Horse, Rabbit, Mouse, Sheep, Pig.

Detection Methods

  • Horseradish peroxidase (HRP) with chemiluminescent substrate.
  • Light detected by photographic film or camera.