Method # 1: Absorbance of DNA

Method # 2: DNA micro-chip technology

Method # 3: Gel Electrophoresis
 
 

The discussion will still follow (kind of) the outline from the text:

5.7 The Isolation & Characterisation of
        Particular DNA Fragments

5.8 The Polymerase Chain Reaction

5.9 Determination of the Sequence of
        Bases in DNA

First, a few words about the UV Absorbance of DNA, and how you can easily monitor whether the DNA is double-stranded or single-stranded (or even triple-stranded for that matter, but this is a topic for another lecture...)

This is kind of an ugly picture of double-stranded DNA (scanned in from my lab notebook), but you can see that the peak is very close to 260 nm. (Actually, very pure DNA should have a lambda max. around 257 nm.)
 

How do I know what the concentration is?
 
 

DNA Absorbance and melting of the double strand

The Absorbance of DNA increases as it melts

Figure 5.28, page 200 in Hartl & Jones

Different organisms have different G+C contents...

 

This difference in G+C can be seen in the densities of different DNA:

And the %G+C is also seen by different melting curves:

For Bacteria, the melting curves are all pretty much the same, except for the difference due to G+C content.  However, the melting curves for eukaryotes can be quite different, as can be seen by a comparison of E.coli DNA with DNA extracted from a cow.

This difference in melting curves (actually, renaturation curves) is due to the presence of repetative sequences within the genome of cow DNA.  By melting the DNA and then measuring the rate of renaturation, it is possible to estimate the complexity of the genome for the cow (or whatever other organism you want...)
 

.

 

This analysis was extended to many different types of organisms, with the following (now famous) result:

Figure 6.17 (page 238 from Hartl & Jones)

 

Method # 2: DNA micro-chip technology
 
Recently, small DNA oligomers have been attached to micro-chips, and used to probe for the presence of complimentary DNA in bacteria by hybridization.  Here is an abstract for a recent article.

Nat Biotechnol 1998 Jan;16(1):45-48  Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.  de Saizieu A, Certa U, Warrington J, Gray C, Keck W, Mous J  Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.    We have used high-density oligonucleotide probe arrays (chips) for bacterial  transcript imaging. We designed a chip containing probes representing 106  Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The  apparent lack of polyadenylated transcripts excludes enrichment of mRNA by  affinity purification and we thus used total, chemically biotinylated RNA as  hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of  selected genes. This technology allows simultaneous and quantitative measurement  of the transcriptional activity of entire bacterial genomes on a single  oligonucleotide probe array.  PMID: 9447592, UI: 98108853

 

These arrays have been postulated to be capable of analysis of complete genomes:
 

Photolithographic techniques inspired by the semiconductor industry are the basis for preparing high-density oligonucleotide arrays. Shown here is a 1.28x1.28­cm array of more than 10,000 different nucleotide sequences (probes), which was then incubated with a cloned fragment (the target) from the genome of the HIV-1 virus. If the fluorescently labeled target contained a region complementary to a sequence in the array, the target hybridized with the probe, the extent of the hybridization depending on the extent of the match. This false-color image depicts different levels of detected fluorescence from the bound target fragments. Techniques such as this may ultimately be used in sequencing applications, as well as in exploring genetic diversity, probing for mutations, and detecting specific pathogens. Photo courtesy of Affymetrix.   Link to the page this came from:  http://www.lbl.gov/Publications/TKO/07_beyond.html

How can they possibly make such a fine grid with DNA oligomers?

One way is to use a GRIDDER robot, such as this one from the Sanger centre in England (that's why "center" is spelt funny!)
 
 

Gridding on the Flexible Robot   What Is It...?  The gridder makes high density arrays (grids) of spots onto nylon membrane filters. These spots can be cosmid, YAC, BAC or DNA. High density arrays mean that several microtitre plates can be represented on a single 80mm x 128mm filter, e.g. with a 4x4 pattern 16 plates are condensed onto a single filter.  (I guess I should give a few definitions, since we haven't covered this yet in lecture -  a cosmid is a large piece of viral DNA, YAC = Yeast Artificial Chromosomes;  BAC = Bacterial Artificial Chromosomes)

Here is an extract of a 6x6 pattern using a 96-pin tool. The pattern only contains 32 spots rather than the maximum 36, the missing spots are used to make the pattern easier to read.

How Does It Do It...? The gridder is built on the Flexible Robot platform. The set up looks something like this:     Here you can see the 96-pin tool on the end of the Z axis and two freezer trays on the bed of the robot, one holding master plates, the other holding output filters. To the right of the freezer trays is a fixture holding an ethanol bath for sterilisation.

The gridder uses a tool comprising 96 or 384 floating pins. These are arranged on 9mm or 4.5mm centres to suit standard plasticware. The pins are guided by two PTFE loaded nylon guide plates to provide the accurate guiding and low friction needed to produce high quality, high density patterns.  This is what it looks like...you can see the 96 floating pins and the filters.

Where Can I Get One...? If you're from the Sanger Centre or any of the other institutions on the Hinxton Hall site then come down to the Engineering Group in the workshop.  If you're looking at this from a site other than Hinxton Hall then contact PBA Technology via their web page.

The Engineering Group home page   The Sanger Centre home page  Rob Davies (rmd@sanger.ac.uk) Last Update 16 July 1996

 
 
 

This technology has now allowed for the rapid evolutionary analysis within different organisms:
 

Volume 18 Number 2 - February 1998  letters  Evolutionary sequence comparisons using high-density oligonucleotide arrays   Joseph G. Hacia1, Wojciech Makalowski2, Keith Edgemon1, Michael R. Erdos1, Christiane M. Robbins1, Stephen P. A. Fodor3, Lawrence C. Brody1 & Francis S. Collins1  1National Human Genome Research Institute, Building 49/3A14, National Institutes of Health, Bethesda, Maryland 20892, USA. 2National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA. 3Affymetrix, 3380 Central Expressway, Santa Clara, California 95051, USA. Correspondence should be addressed to F.S.C. e-mail: fc23a@nih.gov   We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes.          Copyright 1997 Nature America Inc. Send comments to webmaster@us.nature.com

Here's an advertisement for a company, using DNA chip technology:
 

High Throughput Gene Expression Analysis:  Customized screening for expression levels of up to 100,000 genes on high density arrays, using diseased tissues, normal tissues and cell lines.   click here for a link to Lifespans homepage

 

Method # 3: Gel Electrophoresis

Gel electrophoresis allows for the seperation of DNA molecules based on their size.

Restriction Enzymes

Restriction enzymes cut DNA at specific sequences.  Here's a few examples...

Restriction mapping:

these enzymes can be used to map a given DNA sequence

Southern Blots

note that you can also use fluorescently labelled DNA - it does not HAVE
to be radioactively labelled!

 note that your DNA is a smear, because you have lots of different fragments - you want to only see a few bands (hopefully) with your probe.

To transfer the DNA from the gel to the membrane, usually a "sandwich" is made as follows:

 

The gel is usually then thrown away.  I like to use pre-stained markers, so I can make sure that the DNA transfers to the filter.

 

The "Seal-a-Meal" bag is not essential, but you certainly want to be careful not to leak radioactive stuff all over the lab!!  Sometimes the filter will be put in a "roller bottle" and then hybridised in an oven.  I think the oven kind of looks like a "hot dog cooker".

FINALLY, you remove the liquid containing the probe, and (carefully) wash the membrane.  Hopefully you'll see some bands on the film (next step).

 

Usually the membrane is exposed to film overnight or so....

 

Here is a typical example of a Southern blot.  YES, it looks ugly.  Most of the time they look this ugly or worse!  But, the important thing is that you can see your bands, so to the poor person who did this experiment, it probably looks "beautiful"!

 

5.8 The Polymerase Chain Reaction

Figure 5.36 in Hartl & Jones, 1998 (page 209).

 

There are may different types of PCR, including the use of reverse transcriptase to make a DNA from an RNA molecule, ("RT-PCR") but these will be the topics of future discussion....

5.9 Determination of the Sequence of
        Bases in DNA

Here is a EXCELLENT link for

Presently there are three - no, FOUR (maybe FIVE) methods for DNA sequencing:

1. Maxam & Gilbert's method (chemical cleavage)

2. Fred Sanger's method (dideoxy method)

3. AUTOMATED sequencing (dideoxy, using fluorescent tags)

4. DNA-chip sequencing (currently being developed)

5. Sequencing of individual bases (at the atomic level!)

    The advances in methodologies can be seen in the increase in sequencing power.  The human genome is roughly 3,000,000,000 bp long.  Below is a table where I have very roughly estimated the length of time it would take to sequence the human genome, based on the number of bp that have been sequenced in a given year.
 

Link to a more recent list of sequenced genomes

Link to lecture notes from Autumn 1999

Now a bit more about each method of sequencing:

1. Maxam & Gilbert's method (chemical cleavage)

Yes, the Maxam Gilbert gels often will look this ugly!

2. Fred Sanger's method (dideoxy method)

Click here for a sample protocol for CHEMILUMINESCENT DNA SEQUENCING

3. AUTOMATED sequencing (dideoxy, using fluorescent tags)
 

4. DNA-chip sequencing (currently being developed)

Genome Res 1997 Jun;7(6):606-614  Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.  Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvanen AC  Department of Human Molecular Genetics, National Public Health Institute,  Helsinki, Finland.  We describe a method for multiplex detection of mutations in which the  solid-phase minisequencing principle is applied to an oligonucleotide array format.  The mutations are detected by extending immobilized primers that anneal to their  template sequences immediately adjacent to the mutant nucleotide positions with  single labeled dideoxynucleoside triphosphates using a DNA polymerase. The  arrays were prepared by coupling one primer per mutation to be detected on a  small glass area. Genomic fragments spanning nine disease mutations, which were  selected as targets for the assay, were amplified in multiplex PCR reactions and  used as templates for the minisequencing reactions on the primer array. The  genotypes of homozygous and heterozygous genomic DNA samples were  unequivocally defined at each analyzed nucleotide position by the highly specific  primer extension reaction. In a comparison to hybridization with immobilized  allele-specific probes in the same assay format, the power of discrimination  between homozygous and heterozygous genotypes was one order of magnitude  higher using the minisequencing method. Therefore, single-nucleotide primer  extension is a promising principle for future high-throughput mutation detection  and genotyping using high density DNA-chip technology.  PMID: 9199933, UI: 97343328

5. Sequencing of individual bases (at the atomic level!)
 

Approach # 1 - use of Scanning-tunneling Electron Microscopy.
This method is currently being developed in the lab of Carlos Bustamante

 

Approach # 2 - fluorescence energy transfer from individual
                           DNA bases

Sequencing based on the detection of fluorescence from single molecules is being pursued at Los Alamos. The strand of DNA to be sequenced is replicated using nucleotides linked to a fluorescent tag -- a different tag for each of the four nucleotides. The tagged strand is then attached to a polystyrene bead suspended in a flowing stream of water, and the nucleotides are enzymatically detached, one at a time. Laser-excited fluorescence then yields the nucleotide sequence, base by base. Much development remains to be done on this technique, but success promises a cheaper, faster approach to sequencing, one that might be applicable to intact cosmid clones 40,000 bases long.

  Alaskan Victim of 1918 Flu Yields Sample of Killer Virus (11-Feb-98)

Last modified on: 1 February, 2000 by Dave Ussery