Arizona State University College of Liberal Arts and Sciences

Bio343

Chemical Synthesis, Sequencing, and Amplification of DNA

(Chapter 5)


DNA sequencing

With the development of automated DNA sequencers any sequence can be obtained readily and rapidly. However, it should be kept in mind that cloning and DNA isolation (required for DNA sequencing by any means) is also labor-intensive. Automation at that level is available as well, but remains expensive. Larger sequencing laboratories (such as those involved in genomic sequencing efforts) employ automation at the DNA sequencing, sample preparation, and interpretational levels, and the amount of new sequence information that has become available over the past few years has grown almost exponentially. The science of interpreting DNA sequence information and accessing it in ways that are as efficient as possible is named bioinformatics.

Genomic sequencing currently is carried out by breaking the genome into small pieces (1,000-3,000 nucleotides), cloning them, and sequencing each piece individually. The entire sequence is then put together by overlapping the sequences of all pieces. Particularly for larger genomes the pasting together is challenging in view of the occurrence of repeats in the sequence, but with sufficient controls a reliable result can be obtained. Currently, a large laboratory specializing in sequencing (for example, http://genome.jgi-psf.org/mic_home.html) can obtain a complete genomic sequence of a bacterium in just a day or so. However, functional assignment of genes etc. takes much longer as this requires brain power and sometimes detailed experimentation for verification.

Polymerase Chain Reaction (PCR)

PCR has been summarized at http://web.mit.edu/esgbio/www/rdna/pcr.html and at http://www.accessexcellence.org/AB/GG/polymerase.html, and a page about Kary Mullis, the presumed "inventor" of PCR for which he has received the Nobel Prize, can be found at http://www.accessexcellence.org/AB/BC/Kary_B_Mullis.html.

The practical applications of PCR are quite amazing. For example, with this method sufficient DNA can be prepared from single hairs or from blood stains to do Southern blots or DNA sequencing. This of course is of importance for forensic applications. For paleontologists and archeologists, the application is a little different: Sufficient DNA can be isolated from a mammoth that froze to death in Siberia many millennia ago, from a Bronze-Age hunter found recently in the ice of a glacier in the Alps, from mummies in Egypt, from 8,000-year-old human brain tissue, from magnolia leaves encapsulated in Miocene shale, or from 11,000-year-old bison bones, to amplify to quantities sufficient for sequencing. However, the amount of DNA needed for sequencing is becoming smaller: In 2005, rather degenerated DNA isolated from a long extinct cave bear was sequenced without amplification. The record of DNA recovery from ancient organisms currently is 40 million years: reasonably intact DNA has been recovered from a termite that was embedded in amber some two dozen million years after the dinosaurs became extinct. From sequencing of specific genes (for example, rRNA genes), evolutionary relationships between the old (sub)species and current representatives can be deduced. Even more reminescent of "Jurassic Park", ancient bacterial spores from the intestines of a 25-40 million year old bee that was preserved in amber reportedly have been revived and cultured. The resulting bacterial culture is most closely related to Bacillus sphaericus.

An interesting PCR application involves the identification of the remains of the Romanovs, the Russian czar family killed in 1918 by soldiers of the Bolshevik army as ordered by Stalin. The killing of czar Nicholas II and his family is one of the haunting tales of the Russian Revolution. According to "legend", the bodies of the czar family and that of servants and the family doctor were buried in a shallow road-side grave near Jekatarinenburg (formerly Sverdlovsk) after the truck on which their corpses were carried broke down; according to the same "legend", concentrated sulfuric acid was poured over the corpses to make positive identification impossible (or so they thought), and bones were broken and scattered through the grave. After the end of the Soviet era, the location of the putative grave was disclosed, and indeed some badly decomposed and partially broken human bones were found. But how can one positively identify this grave as belonging to the czar and his household? Best start with bone (that's all they had to go by anyway). One gram of sulfuric acid-treated bone may yield some 10-50 pg (1 pg = 10-12 g) of DNA, enough to do PCR. Both tandem-repeat chromosomal and hypervariable mitochondrial sequences were amplified. Note that mitochondrial DNA is inherited maternally only. Sex determination of bones was done by amplification of the gene for amelogenin that is common to X and Y chromosomes, and that yields different PCR sizes depending on whether it is on an X or a Y chromosome.

A key player in the positive identification of the bones as belonging to the czar family was Prince Philip, husband of the British Queen. He is the great-grandson, by maternal lineage, of the mother of the czar's spouse. The mitochondrial DNA sequence of Prince Philip matched up with DNA from four reconstructed skeletons, one of an adult and three of children, but not with any of the other skeletons. This implies the adult skeleton is that of the czar's spouse, and the other three belong to three of her five children. Comparing PCR sequences of nuclear DNA from the children's bones with that of the remaining skeletons in the grave, the czar's skeleton could be identified. Also, four of the nine skeletons are not related to each other or to one of the other five. These four skeletons presumably are of three servants and of the family doctor. The skeletons of two of the children, presumably those of Alexei (the tsarevitz) and Anastasia, one of the daughters, were not in the grave. They either survived (quite a few contenders to being Alexei or Anastasia have come and gone over the years; one of them died some years ago in Scottsdale) or were burned and buried separately. With the current knowledge on the nuclear and mitochondrial genome of the czar's family, it is easy to develop a DNA fingerprint of the missing children. So far, none of the claims have proven to be true; however, any proven heirs may await what is left of the family fortune (if Russia does not succeed in its claim to these funds).

Question, Chapter 5

According to science fiction stories such as in "Jurassic Park" it would seem that one could regenerate a dinosaur from some ancient genetic material. What are the specific criteria that would need to be met, in principle, in order to be able to amplify parts of an ancient genome and to piece them together? Do you think this provides some practical problems?


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Instructors | Aims
Lecture Part: Schedule | Expected Background & Textbook Info | Historical Perspective
Intro to Biotechnology | DNA, RNA and Protein Synthesis | Chemical Synthesis, Sequencing, and Amplification of DNA |
Directed Mutagenesis and Protein Engineering | Vaccines | Antibiotics & Proteins | Bioremediation |
Microbial Insecticides | Plant Genetic Engineering: Methodology | Plant Genetic Engineering: Applications | Transgenic Animals
Human Molecular Genetics | Regulatory & Ethical Aspects | Biotech Inventions | Additional Materials
Lab Part: Aims and Expectations | Schedule

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