Introduction to the “Family Tree” of DNA Methods

dna-method-overview-v10

Many methods in molecular biology are simply different combinations of a handful of techniques. These combinations can be represented as a “family tree” whose “trunk”/”backbone” is PCR (polymerase chain reaction) and whose “roots”/”foundation” is built upon: (1) chemical synthesis of short oligonucleotides, (2) fully sequenced genomes and (3) vectors derived from bacteria and viruses.

DNA/RNA-BASED PROBES:

Even without PCR, these foundational technologies are sufficient to produce several short, anti-sense DNA/RNA probes including:

  • Fluorescent probes: for microscopy and histology
  • short interfering RNA: for gene knockdown by RNA interference
  • Microarrays: for transcriptome analysis.

Due to the limits of solid-phase DNA synthesis (oligo-size < 200 kbp), PCR - which can produce oligonucleotides up to 20kbp in size - forms the backbone of most DNA based techniques described below.

QUANTITATIVE PCR FOR CONCENTRATION MEASUREMENT:

PCR makes and purifies specific DNA segments from genomic DNA, using gene-specific primer sets (). Quantitative PCR methods, uses fluorescent probes to track the PCR-amplification time course and directly measure DNA copy number (qPCR) or RNA expression level (RT-PCR). A few applications of quantitative PCR methods are listed below:

  • qPCR (DNA): gene copy number, transgene copy number for GMO’s, plasmid copy number, % viral dna incorporation, bacterial/viral load per gram tissue,
  • RT-PCR (DNA): mRNA expression (often a surrogate for protein expression), non-coding RNA expression, RNA viral load

AGAROSE GELS FOR STRUCTURAL QUESTIONS:

Agarose Gels are the primary means to study the structure of DNA and RNA that has been made and purified by PCR. These gels separate DNA and RNA by weight/mass and can be used to determine purity and identity or PCR products. In addition, analysis of fragmentation patterns (using nucleases, dideoxy-bases, etc.) can be used to study the primary structure (sequencing), secondary structure (DNA and RNA folding) and molecular interactions of purified DNA and RNA.

CLONING FOR SYNTHETIC DNA-BASED TOOLS:

Finally, unnatural primers can be used to add/change features of genomic DNA including the addition of: (1) mutations, (2) restriction sites and (3) barcodes. The addition of mutations and restriction sites are very important for the development of cloning tools which combine PCR-amplified DNA with a self-replicating bacterial or viral vector. Some of the most important cloning tools include

  • Transcriptional clones: enable large-scale amplification of DNA within cells
  • Expression clones: enable large-scale protein production and mutagenesis within cells
  • Reporter clones: enable the production of cell-based fluorescent/colorimetric probes that are useful for studying signalling or high-throughput screening

 

REFERENCES:

  1. Beaucage, S.L.; Iyer, R.P. Advanced in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron, 1991, 48, 2223-2311.
  2. Molecular Cloning: Technical Guide v. 2, 2014, New England Biolabs Inc.
  3. Alberts, B. Molecular Biology of the Cell 5th Ed. Garland Science 2008
  4. Murphy, K. Janeway’s Immunobiology 8th Ed. Garland Science 2012

 

Creative Commons License
This work by Eugene Douglass and Chad Miller is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

Leave a Reply

Your email address will not be published. Required fields are marked *

SpamBot Test * Time limit is exhausted. Please reload CAPTCHA.