Nucleic Acids and Proteins Summary

  • Labeling of Nucleic Acids:
    • Used to detect presence, amount, or location of a particular nucleic acid by hybridization.
    • Can determine rate of synthesis or degradation.
    • Precursors contain radioactive isotopes like ^{32}P, ^3H, or ^{14}C, with ^{32}P preferred for its stronger emission and shorter half-life.
    • Labeling with ^{32}P can occur at α, β, or γ positions of NTP or dNTP, but only the α form allows incorporation during polymerization.
  • Labeling of Total Nucleic Acids:
    • Involves using specific precursors like deoxythymidine for DNA and uridine for RNA.
    • Cells are incubated with labeled precursor (pulse) followed by non-labeled precursor (chase).
    • Extraction and quantification determine total replication or transcription rate, but requires comparison between experimental conditions.
  • Radioactive Probe Labeling:
    • Cloned DNA can be labeled in vitro for use as a probe.
    • Achieved by incorporating labeled nucleotides or molecules into nucleic acids.
    • High-density labeling, where the entire length is uniformly labeled, is preferred for stronger hybridization signals.
    • Detected by autoradiography after annealing.
  • Uniform Labeling Procedures:
    • Nick Translation:
    • E. coli DNA polymerase I synthesizes labeled DNA using α-^{32}P-dCTP, dNTP, and a buffer.
    • DNase-I introduces single-stranded nicks, and DNA polymerase-I initiates synthesis at the 3' end of nicks, excising nucleotides at the 5' end.
    • Resulting probe is highly radiolabeled, hybridizing to the DNA sequence of interest.
    • Random Priming:
    • dsDNA is denatured & DNA polymerase synthesizes labeled DNA by extending random hexamer primers.
    • Polymerases lacking 5'-3' exonuclease activity (e.g., Klenow fragment) are used.
    • Newly synthesized strand is completely labeled, resulting in high specific activity probes.
    • PCR Probes:
    • Uniformly labeled probes are generated by incorporating a labeled nucleotide during PCR.
    • Large amounts of probe with high label density can be synthesized from little DNA.
  • 5'-Labeling of Nucleic Acids:
    • Achieved via phosphatase/kinase action, useful for techniques needing low-density labeling.
    • Phosphatase removes the 5'-P, followed by T4 Polynucleotide Kinase transferring the γ-phosphate group of ATP to a 5'-OH terminus.
  • Probe Purification:
    • Important to remove non-incorporated radioactive nucleotides using gel filtration chromatography.
    • DNA molecules are excluded from the column while free nucleotides are retarded due to their ability to penetrate the beads.
  • Non-Radioactive Labeling:
    • Utilizes special DNA precursor molecules like digoxigenin-dUTP (DIG-dUTP) instead of ^{32}P-labeled precursors.
    • Detection involves adding an anti-DIG-AP conjugate, visualized using colorimetric or chemiluminescent substrates.
  • RNA Probes:
    • cDNA of interest is inserted at MCS between SP6 and T7 or T3 promoters and transcribed in vitro (run-off) into ss sense or anti-sense RNA with labeled NTPs.
    • RNA probes produce stronger signals in hybridization reactions due to high specific activity.
  • Sequencing Nucleic Acids: Principle and Aim:
    • Determines the primary structure of nucleic acids, crucial for cloned DNA.
    • Methods use gel electrophoresis to separate DNA fragments of different sizes.
    • Generates DNA molecules with a common 5' end and a base-dependent 3' end.
  • Sequencing Methods:
    • Sanger (enzymatic) method:
    • Uses DNA polymerase to incorporate a 2'-3'-dideoxynucleotide (ddNTP), terminating chain elongation.
    • Four separate reactions with all dNTPs and one ddNTP each.
    • Random chain termination generates fragments of different lengths, visualized by autoradiography after electrophoresis.
  • Electrophoresis and Autoradiography:
    • Sequencing products are sorted in four lanes (A, T, C, G) by electrophoresis on a denaturing polyacrylamide gel, followed by autoradiography.
  • Automated Sequencing:
    • Uses fluorescently labeled nucleotides for automation.
    • Four enzymatic sequencing reactions in one tube, sorted by electrophoresis on a polyacrylamide gel.
    • Capillary electrophoresis (CE) is used for faster separation, with laser-induced fluorescence for detection.
  • RNA Sequencing:
    • Similar techniques to DNA sequencing, but requires reverse transcriptase.
  • Genome Sequencing:
    • Launched in 1986, aimed to sequence the entire human genome and genomes of model organisms.
    • Two approaches: sequencing fragments from BAC clones and shotgun sequencing.
    • Revealed the number of human genes is between 30,000 and 40,000, introns account for 25%, and repetitive sequences for 60%.
  • Sequence Databases:
    • Organized in databases like GenBank and EMBL.
    • Contain information about DNA, RNA, and protein sequences, protein structure, and genetic maps.
    • Facilitated by bioinformatics, which handles biological data in terms of organization, classification, storing, retrieval, comparison, and prediction.
  • Detection of Nucleic Acids by Hybridization- Principle:
    • DNA-DNA and RNA-DNA hybridization is used to detect specific nucleic acid sequences in complex mixtures.
    • Hybridization involves nucleation (formation of short hybrids) and zippering (extension of the hybrid).
    • Membrane hybridization is used, where DNA or RNA is immobilized on a support for hybridization with labeled probes.
  • Southern Blotting:
    • Combines agarose gel electrophoresis with DNA-DNA or DNA-RNA hybridization.
    • DNA fragments are transferred, denatured, and immobilized for hybridization with labeled probes.
  • Southern Blotting: Technique:
    • DNA extraction - digestion - electrophoresis, followed by transfer to a membrane.
    • Upward and downward capillary transfer methods are used.
    • Nucleic acids are fixed on the membrane by drying, alkaline treatment, or UV irradiation.
  • Southern Blotting: Hybridization:
    • Membrane hybridized with a specific labeled probe.
    • Pre-hybridization is performed to saturate the membrane.
    • Stringency (temperature, saline concentration) is crucial for hybridization.
  • Southern Blotting: Autoradiography:
    • Membrane is applied against an X-ray film, revealing bands where probe molecules are bound.
  • Southern Blotting: Applications - Diagnosis:
    • Used for diagnosis of defective genes (genetic diseases) and infectious diseases.
    • Can detect pathogen nucleic acid at a late infection stage or earlier by combining PCR and Southern blotting.
  • Southern Blotting: RFLP in Diagnosis:
    • Uses the difference of restriction map between normal and mutant alleles.
    • A mutation may cause change of restriction map by creating or removing a restriction site.
  • Southern Blotting: DNA Fingerprints:
    • Individual organisms have unique genetic makeup.
    • DNA fingerprinting uses RFLP (restriction fragment length polymorphism) and RAPD (randomly amplified polymorphic DNA).
  • Northern Blotting:
    • Primarily used for determination of steady-state level of RNA transcripts.
    • Useful for monitoring gene expression and assessing RNA stability.
    • Total RNA molecules or mRNA are extracted and sorted by electrophoresis in a denaturing agarose gel.
  • RT-PCR alternative to Northern:
    • Analysis of mRNA expression by a semi-quantitative PCR particularly useful when Northern blot cannot be applied because of low abundance of mRNAs.
  • Northern Blotting: Nuclease S1 (RNAse) protection assay alternative:
    • Based on the ability of Nuclease S1 to digest single stranded RNA.
  • Dot Blot (Slot Blot):
    • Used for determination of RNA relative amount but not the size of the RNA.
  • In Situ Hybridization:
    • Determines spatial distribution of DNA or RNA within cells.
    • Radioactive (RISH) or fluorescence (FISH) in situ hybridization methods are used.
  • Western Blotting:
    • Determines the relative amount of a particular protein and its size.
    • Proteins are sorted by SDS-PAGE, transferred to a membrane, and detected using specific antibodies.
  • Western Blotting Techinique:
    • During SDS-PAGE, proteins move through the gel based on molecular weight.
    • Membrane saturation with non-specific and incubated with a primary antibody.
  • Transcription Initiation Site:
    • Protection against nuclease S1: A labelled template strand is hybridized with RNA, treated with nuclease S1, and run on gel.
    • Primer extension: A labelled primer is designed and lengthened by reverse transcriptase for gel analysis.
  • In Vitro Transcription: Run-on:
    • Aims to study effect of agents on transcription.
    • Soft lysis of cells & nuclei are incubated with labelled nucleotides for RNA molecule incorporation.
  • In Vitro Transcription: Run-off:
    • Total RNA is loaded on membrane & the radioactive RNA sample that was extracted from nuclei is hybridized with corresponding RNA.
  • RNA and Protein Stability:
    • Cells are treated with transcription (actinomycin D) or translation (cycloheximide) inhibitors & is determined by RNA & protein samples.
  • Investigation of Translation Rate:
    • Pulse-chase with 14C-Leucine to study protein modification rate and is tested by Immunoprecipitation by centrifugation steps during Immunoblot.
  • DNA-Protein Interaction:
    • Methods = DNA footprinting (DNase I protection) & EMSA (gel retardation).
  • Footprinting:
    • Determines cloned DNA sites involved in protein interaction by assessing DNA's resistance to DNase I.
  • Gel Shift (EMSA):
    • Detects protein binding to single oligonucleotides based on slowed electrophoresis mobility.
  • Southwestern Blot:
    • Detects protein-DNA interactions by applying labelled oligonucleotide from sorting into EMSA.
  • DNA Curvature:
    • EMSA can monitor DNA conformational changes due to protein binding.
  • DNA Chips - Aims and Principles:
    • High-throughput technology to monitor expression profiles of thousands of genes simultaneously.
    • Based on hybridization of nucleic acids.
  • Diversity of Arrays:
    • Arrays are orderly arrangements of samples (nucleic acid, proteins, antibodies, chemicals,…).
    • Arrays are described as macroarrays or microarrays, depending on the size of the sample spots.
  • Hybridization and Scanning:
    • Allows rapid measurement and visualization of differential expression between genes at the whole genome scale.
    • RNA samples are extracted & the slide scanning allows analysis of gene induction.
  • Applications: Genotyping:
    • DNA sequence polymorphisms through chromosome analysis & hybridization signals are then analyzed.
  • Protein Arrays:
    • Used for proteomic research by spotting on silicon chips where antibodies are added, enabling determination of protein's amount.