This is the first part of my Master's degree thesis. The entire thesis is available for download in word format by clicking here. I thought I'd post it here in case anyone wanted to know the type of work that I do for a living.
Evaluation of Random Amplified Polymorphic DNA Genetic Markers in the Identification of Hypertension Related Loci in Inbred Dahl Rats.
by
Christopher Andrew Baba
A Graduate Project submitted to The Johns Hopkins University
in conformity with the requirements for
the degree of Master of Science in Interdisciplinary Science Studies
Baltimore, Maryland
Spring of 1996
Faculty Sponsor
John Hamlyn Ph.D.
Department of Physiology
University of Maryland at Baltimore
Abstract
Hypertension is a common disorder in Western acculturated societies whose etiology is unknown. Two strains of inbred rats have been developed that may be a good model of human essential hypertension. One strain develops arterial hypertension if given a high salt diet (sensitive), while the other strain does not (resistant). One possible strategy to determine the genes responsible for this form of hypertension is to cross the two strains and correlate the blood pressure of the F2 offspring with the inheritance of genetic markers from the parental strains. However, few genetic markers have been mapped in rats. I investigated the feasibility of random amplified polymorphic DNA to generate a large number of genetic markers in these rats. A computer program was developed to analyze genetic marker data and was tested using simulated data. The results of these simulations showed that the general location of the gene(s) responsible for salt-sensitive hypertension could be detected using as few as one hundred rats and 250 markers. This method would require 25,000 RAPD-PCR reactions, 100 of which could be processed per day by a skilled technician. Approximately 250 technician days would be required to generate enough markers for statistically significant results. This thesis indicates that the application of random amplified polymorphic DNA is a convenient, cost-effective, and rapid means to locate specific genomic regions mediating complex polygenic traits in laboratory animals.
Introduction
In the 1960's, Lewis Dahl and coworkers developed an animal model of salt-sensitive hypertension. Outbred strains of Sprague-Dawley rats were selectively bred for sensitivity (SS) or resistance (SR) to hypertension induced by high salt diets1. Fully inbred strains of these rats were later developed by John Rapp by repeated brother-sister matings2. The mechanisms that link salt with hypertension in these rats are largely unknown, but experimental evidence points to the kidneys as the major contributor of this effect.
No comprehensive effort has yet been made to use chromosomal mapping to study the genetic linkage between hypertensive loci and genetic markers in inbred Dahl rats. This has not been completed due to the enormous cost and effort of obtaining hundreds of genetic markers from hundreds of rats. Another problem has been that accurate phenotyping of rat blood pressure is not always possible due to non-genetic influences such as environmental variation.
Some success has been reported in the genetic mapping of the stroke-prone hypertensive rat, a strain of rat which develops hypertension spontaneously4,5,6,7. There are also reports of several genes that may cosegregate with blood pressure in Dahl rats, but these were done by screening several candidate genes rather than whole genome mapping8,9.
A new method of obtaining genetic markers using the polymerase chain reaction (PCR) and arbitrary primers was recently described by Williams et. al3,10. Known as Random Amplified Polymorphic DNA (RAPD) this method appears to be quicker and less labor intensive than the previously used methods such as Restriction Fragment Length Polymorphism (RFLP) analysis. The RAPD method uses arbitrarily defined 10-mer oligonucleotides as PCR primers and genomic DNA from the rats as the template. Short segments of DNA that lie between the primers are amplified by the PCR process, then the amplified products are separated by agarose gel electrophoresis (see Figure 1). This usually yields 5 to 10 bands of different sizes each one corresponding to a single locus in the genome. Theoretically there is no limit to the number of primers used to obtain markers (up to the 1,048,576 nucleotide combinations in a 10-mer) so any desired number of markers can be obtained. Analysis of parental and F1 and F2 generations by the RAPD method can lead to genomic linkage maps. The chromosomes and the arms on which these markers lie can be determined by a comparison to RFLP markers or to other known genes.
Recent advances in mathematical models of Mendelian inheritance of quantitative traits11 (such as blood pressure), and the development of computer algorithms to analyze large numbers of genetic markers, provide a secure theoretical foundation on which to base data analysis.
The main goal of this project was to evaluate the technical suitability as well as the feasibility of using the RAPD method to elucidate the locus or loci of hypertension related genes using inbred salt sensitive (SS) and salt resistant (SR) rats of the Dahl strain.
Specific goals were to:
1) Optimize the PCR reaction and other lab protocols for best reproducibility.
2) Determine and quantitate the genetic differences between the SS and the SR rats. (If they are too closely related it will be harder to find markers unique to each strain of rats.)
Figure 1. The Random
Amplified Polymorphic DNA (RAPD) method works by amplifying short segments of
DNA located between arbitrarily defined primers. Each band is associated with a
single locus in the genome and can be used as a genetic marker to trace
inheritance from parents to offspring. Association of a given phenotype with a
marker is good evidence that the genes governing that phenotype are located near
the marker.
3) Find or develop an appropriate method for data analysis.
4) Theoretically examine the problems that arise if hypertension is controlled by more than one locus in these rats, and the effect that would have on analyzing results. This would be done by evaluating different scenarios, using simulated data, by varying the number of loci and the contribution each has on blood pressure, and determining the number of rats and markers that would be required for statistically significant results under each scenario.
5) Theoretically examine the problems introduced by non-genetic factors on blood pressure, such as errors and environmental variation, and the effects they would have on analyzing results. This would be done by: a) evaluating different scenarios, using simulated data, and by changing the magnitude of error typically associated with laboratory measurements b) looking at published data on blood pressure variation between Dahl SS and SR rats to determine a most likely scenario c) determine the number of rats and markers that would be required for statistically significant results under each scenario.
6) Evaluate different scenarios that have defined errors and multiple loci, and under these conditions, determine the number of rats and markers that would be required for statistically significant results.
I had anticipated using software already developed for the analysis of quantitative trait loci (QTL), such as the "Mapmaker-QTL" program12 for most of this project. However, at the early stages of this thesis, it was apparent that a better means of analyzing QTL data might be feasible, and I wrote my own software using Microsoft's Excel's Visual Basic for Applications programming language13. The theory behind this software is described in the materials and methods section, and the computer code is printed in the appendix. A floppy disk of the computer program and associated spreadsheets is also enclosed.
The entire thesis is available for download in word format by clicking here.