DNA fingerprinting

Examples of DNA fingerprinting in the following topics:

• Classification of Microorganisms

  • Classification of microorganisms has been largely aided by studies of fossils and recently by DNA sequencing.
  • The most widely employed methods for classifying microbes are morphological characteristics, differential staining, biochemical testing, DNA fingerprinting or DNA base composition, polymerase chain reaction, and DNA chips.

• DNA Analysis Using Genetic Probes and PCR

  • Hybridization of the sequence with a complementary sequence of DNA or RNA, follows cleavage of the double-stranded DNA of the microorganism in the specimen.
  • At the national or international level, fingerprinting allows strains from different geographic areas to be compared, and the movement of individual strains to be tracked.
  • Fingerprinting technique requires high-quality genomic DNA, which is not only difficult to prepare but also requires culturing of the organism, resulting in a long turnaround time.
  • In addition, fingerprint interpretation and matching can be complicated and require sophisticated computer software for large-scale analysis.
  • However, spoligotyping has significantly less discriminatory power than fingerprinting.

• Amplifying DNA: The Polymerase Chain Reaction

  • DNA cloning for sequencing; DNA-based phylogeny, or functional analysis of genes
  • The identification of genetic fingerprints (used in forensic sciences and paternity testing)
  • PCR is used to amplify a specific region of a DNA strand (the DNA target).
  • DNA template that contains the DNA region (target) to be amplified
  • The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

• DNA Protection Analysis

  • DNA protection or "footprinting" analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction.
  • DNA protection or footprinting is a technique from molecular biology/biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage.
  • This makes it possible to locate a protein binding site on a particular DNA molecule.
  • The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein.
  • If the protein binds DNA, the binding site is protected from enzymatic cleavage.

• Whole-Genome DNA-Binding Analysis

  • Whole-genome DNA-binding analysis is a powerful tool for analyzing epigenetic modifications and DNA sequences bound to regulatory proteins.
  • After cell lysis, the DNA is fragmented by sonication.
  • DNA bound by the protein will be coprecipitated and enriched, compared to DNA not bound by the respective protein.
  • Two different fluorescence labels are used to label the IP DNA, and a hybridization-control DNA, respectively.
  • Usually, total DNA before IP (input DNA) is used as hybridization control.

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation .
  • Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis.
  • The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.
  • Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day.
  • The four DNA bases are represented by different colours which are interpreted by the software to give the DNA sequence above.

• Obtaining DNA

  • When cloning genomic DNA, the DNA to be cloned is extracted from the organism of interest.
  • For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest.
  • DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene synthesis). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer.
  • Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme.
  • The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

• Supercoiling

  • DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on that strand.
  • DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on that strand .
  • DNA supercoiling is important for DNA packaging within all cells.
  • Supercoiling of DNA reduces the space and allows for much more DNA to be packaged.
  • Because DNA must be unwound for DNA and RNA polymerase action, supercoils will result.

• Recombinant DNA Technology

  • Recombinant DNA technology also referred to as molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of a specific DNA sequence.
  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI.
  • For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest.
  • DNA of interest is ligated into a vector.

• Molecular Products from Microbes

  • These enzymes have the ability to cut DNA at specific recognition sequences and have served as invaluable tools in DNA modification and manipulation.
  • DNA ligase plays a key role in molecular biology processes due to its ability to insert DNA fragments into plasmids.
  • The process of DNA ligation is defined as the ability of DNA ligase to covalently link, or ligate, fragments of DNA together.
  • In molecular biology -- specifically, during the process of developing recombinant DNA -- DNA ligase can be used to ligate a fragment of DNA into a plasmid vector .
  • The most commonly used DNA ligase is derived from the T4 bacteriophage and is referred to as T4 DNA ligase.