Human Genomic DNA is suitable for:
- Southern hybridization analysis
- genomic library construction
- the amplification of large DNA targets by the Expand System
- to assess the quality or integrity of DNA sample using qPCR or real time (RT) PCR and as a control during DNA sequencing
The amplification of very long fragments of genomic DNA requires template DNA of very high quality. In some cases, amplification failure may be due to poor template quality. This particular quality of genomic DNA is prepared to ensure reliable amplification of long DNA fragments.
Function test: The preparation is used as template in a PCR with the Expand PCR System and appropriate primers from the human tPA Control Primer Set. Amplification products up to 27kb long are obtained.
Absence of contaminating organisms: The serum used for this preparation was tested for HBs antigen and the presence of antibodies to HIV-1, HIV-2, HCV. All tests were negative.
What is Genomic DNA?
Genomic DNA constitutes the total genetic information of an organism. The genomes of almost all organisms are DNA, the only exceptions being some viruses that have RNA genomes. Genomic DNA molecules are generally large, and in most organisms are organized into DNA–protein complexes called chromosomes.
The size, number of chromosomes, and nature of genomic DNA varies between different organisms (see table Sizes and molecular weights of various genomic DNAs). Viral DNA genomes are relatively small and can be single- or double-stranded, linear, or circular. All other organisms have double-stranded DNA genomes. Bacteria have a single, circular chromosome.
In eukaryotes, most genomic DNA is located within the nucleus (nuclear DNA) as multiple linear chromosomes of different sizes. Eukaryotic cells additionally contain genomic DNA in the mitochondria and, in plants and lower eukaryotes, the chloroplasts. This DNA is usually a circular molecule and is present as multiple copies within these organelles.
Chemical and Synthetic Biology Approaches To Understand Cellular Functions
Preparation of genomic DNA
gDNA must be appropriately fragmented before being transformed into yeast spheroplasts. Previous studies have demonstrated that homologous recombination combining two linear pieces of DNA into a yeast artificial chromosome is much more efficient if the homologous arm sequences are closer to the ends of the targeted DNA fragment (Noskov et al., 2003).
Ideally, site specific double-stand breaks (DSBs) can be introduced directly upstream and downstream of BGCs of interest by restriction enzyme digestion, and homology arm sequences should be designed as close to these restriction sites as possible. However, this approach can be limited due to the lack of appropriate restriction sites directly beyond and not within BGCs of interest. Alternatively, gDNA can be randomly fragmented by shearing with a needle or pipette tip.
Recently, Dr. Natalay Kouprina and colleagues have successfully introduced DSBs to the ends of targeted gDNA fragments using the CRISPR-Cas9 endonuclease in vitro, resulting in high efficiency DNA capture, up to 32% for large fragments isolated from complex gDNA (Lee, Larionov, & Kouprina, 2015). However, in vitro CRISPR/Cas9-mediated DNA digestion necessitates several troubleshooting steps and thus may not be an attractive approach if commercial restriction enzymes can be used.
The quality of the gDNA also greatly affects recombination efficiency. For isolation of gDNA from a single organism in pure culture, standard gDNA isolation procedures or commercial isolation kits are generally adequate, but gDNA should be checked by Nanodrop, Qubit, and agarose gel to confirm integrity. Finally, if the target of interest is low in quantity (such as from metagenomic DNA), PCR and/or genomic library preparation are encouraged for enrichment.
Genome Sequence Databases: Genomic, Construction of Libraries.
- Genomic DNA must be isolated from proteins and other cellular debris prior to any enzymatic or mechanical manipulation. Bacterial cells are lysed, typically through exposure to surfactants, such as sodium dodocyl sulfate or Tween-20, or treatment with lysozyme to digest the polysaccharide component of cellular membranes and proteinase K for protease digestion. DNase-free RNase may be added to the lysis step to minimize RNA contamination.
- Genomic DNA can be purified from cell lysate using a phenol–chloroform extraction followed by an ethanol precipitation or commercially available silica columns. Commercially available kits for genomic DNA isolation are often desirable in that they avoid the use of phenol and chloroform, which are toxic and may interfere with downstream enzymatic reactions. Many commercially available kits avoid phenol by using buffers containing the chaotropic agent guanidine hydrochloride to aid in cell lysis and to effectively denature proteins. Purified genomic DNA should be maintained in a nuclease-free Tris buffer or in nuclease-free water.
- Nuclease contamination is a frequent concern associated with genomic DNA isolation. Nuclease activity will degrade DNA and can be easily mistaken for a restriction enzyme digestion or the result of mechanical shearing. Nuclease contamination may be detected by incubating an aliquot of purified DNA at 37 °C for 18 h and then visualizing the DNA on an agarose gel.
- A control aliquot of DNA that had been stored frozen should be used for comparison. Following electrophoresis, if the incubated aliquot appears to have degraded, nucleases may be contaminating the genomic DNA sample. Additionally, the DNaseAlert kit available from Ambion (Austin, TX) can be used to detect DNase contamination in a sample.
- This kit detects DNase in a sample through the use of modified oligonucleotides that fluoresce upon cleavage by nucleases that may be within the sample. If nuclease activity is detected, the DNA sample can be exposed to high concentrations of guanidine hydrochloride (6–8 M) for 30 min or be subjected to an additional phenol–chloroform extraction for more thorough deproteinization of the sample. An ethanol precipitation should follow the additional deproteinization step to remove the phenol, chloroform, or chaotropic salts.