8: Analyzing DNA

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8.1: Introduction
Agarose is a linear carbohydrate polymer purified from the cell walls of certain species of algae. Agar is a combination of the crude extract that contains agarose and the smaller polysaccharide agaropectin. When dissolved and melted in liquid, agarose strands become tangled together to form a netting that holds the fluid in a gel. Reduction of the fluid creates a higher percentage of the gel that is firmer and contains smaller pores within the netting.
8.2: DNA Replication
The Polymerase Chain Reaction (PCR) is a method of rapidly amplifying or copying a region of DNA in a tube. As the name implies, the technique uses a thermostable DNA Polymerase enzyme to mimic in a tube what happens within a cell during DNA replication. The chain reaction permits us to rapidly copy DNA from very minute source material in an exponential way. This technique is used in forensic science, genetic testing and cloning of rare genes.
8.3: Variable Number Tandem Repeats
The difference in nucleotide sequences between humans lies between 0.1-0.4%. This means that people are greater than 99% similar. But when you look at your classmates around the room, you can see that that small difference amounts to quite a bit of variation within our species. The bulk of these differences aren’t even within the coding sequences of genes but lie outside in regulatory regions that change the expression of those genes.
8.4: Propagating DNA in Bacteria
George Beadle and Edward Tatum first described the concept that each gene corresponded to an enzyme in a metabolic pathway by exposing the yeast Neurospora crassa to mutagenic conditions (Beadle & Tatum, 1941). Following these procedures, Joshua Lederberg continued these studies with Tatum where they generated two mutants strains in Escherichia coli. These bacteria were auxotrophs, unable to generate some basic nutrients necessary to sustain their growth.
8.5: Restriction Enzymes
DNA can be cut by restriction endonucleases (RE). Endonucleases are enzymes that can hydrolyze the nucleic acid polymer by breaking the phosphodiester bond between the phosphate and the pentose on the nucleic acid backbone. Molecular biologists also tend to use these special molecular scissors that recognize palindromes of 6 or 8. By using 6-cutters or 8-cutters, the sequences occur throughout large stretches rarely, but often enough to be of utility.
8.6: DNA Fingerprinting (RFLP)
Restriction fragment length polymorphism (RFLP) is a technique that exploits variations in DNA sequences. DNA from differing sources will have variations or polymorphisms throughout the sequence. Using Restriction Enzymes, these differences in sequences may be teased out. However, if one were to take the entirety of the human genome and chop it up with a restriction enzyme, many indecipherable fragments would be made.
8.7: Cheek Cell DNA Extraction
This page contains instructions on how to extract cheek cells using a cytobrush as well as how to use the PCR beads.
8.8: D1S80 VNTR (Genotyping)
The minisatellite marker D1S80 is located at 1p35-p36. This VNTR is 16 bases long. With a variation of alleles between 3-24 repeats, the locus displays enough diversity to aid in distinguishing between people. Although this is not a CoDIS marker, the use of multiple loci is required to definitively identify samples. The large repeat (16bp) permits the use of standard agarose gel electrophoresis to explore the diversity of this locus in our lab. PCR products range from 430bp to 814bp long.
8.9: DNA Miniprep by Alkaline Lysis (Activity)
Once DNA is introduced and carried in bacteria, we would like to isolate the DNA again for further manipulation. In order to do so, bacteria containing the plasmid of interest is grown in a liquid culture of nutrient-rich broth made of yeast extract called Luria-Bertani Broth (LB). These cultured bacteria are grown until they are of a high concentration overnight. The resulting pellet of bacteria is resuspended in a physiological buffer containing the chelator EDTA.
8.10: Sanger Sequencing of DNA
The polymerization of nucleic acids occurs in a 5′ → 3′ direction. The 5′ position has a phosphate group while the 3′ position of the hexose has a hydroxyl group. Polymerization depends on these 2 functional groups in order for a dehydration synthesis reaction to occur and extend the sugar-phosphate backbone of the nucleic acid. In the 1970s, Fred Sanger’s group discovered a fundamentally new method of ‘reading’ the linear DNA sequence using special bases called chain terminators.
8.11: Next Gen Sequencing
Traditional sequencing of genomes was a long and tedious process that cloned fragments of genomic DNA into plasmids to generate a genomic DNA library (gDNA). These plasmids were individually sequenced using Sanger sequencing methodology and computational was performed to identify overlapping pieces, like a jigsaw puzzle. As technology improved, the cost of sequencing genomes became less expensive. This technology outpaced Moore’s Law, resulting in a dramatic price decrease.