Digitized by the Internet Archive in 2010 with funding from Lyrasis IVIembers and Sloan Foundation http://www.archive.org/details/chromosomeassignOOkays Chromosome Assignment of Human Genes by Ann M. Kays Department of Biology Sweet Briar College April 24, 1996 This senior honors thesis has been awarded high honors. 'd. }<-->^ CL-'V~^ui<^~~- obin Lee Davies, First Reader JudithYalmer, Outside Reader RandolpmMacon Woman's College Mapping Genes to Chromosomes Submitted in partial fulfillment of the requirements for Honors in Biology Ann Kays Sweet Briar College Sweet Briar, Virginia Table of Contents Abstract 1 bitroduction 2 Materials and Methods 12 Results 18 Discussion 23 Conclusion 27 Tables 28 Figures 34 Appendices 49 References 51 Acknowledgments 57 Pledge 58 Abstract Two human genes were assigned to chromosomes. Cartilage-derived morphogenic protein 1 (CDMPl-PEN) was assigned to chromosome 20 and asialoglycoprotein receptor 2 (ASGR2) was assigned to one of six different chromosomes using the polymerase chain reaction (PCR) and gel electrophoresis (Chang, Spiess). PCR is a DNA amplification technique in which a specific sequence is exponentially amplified through the action of a thermostable enzyme that polymerizes complementary nucleotides to a single stranded DNA template to extend primers used to locate the gene by complementary base pairing. Detection is made by analyzing the PCR product using gel electrophoresis, a separation technique in which DNA is caused to migrate through a semi- solid matrix in an electrical current. Chromosomal assignment was made if the primers located the unassigned gene in the chromosomal DNA; exponentially amplified the gene; and produced discemable bands using gel electrophoresis. Data collected in this research project will be beneficial to the Human Genome Project. The Human Genome Project is a world-wide, collaborative research initiative to produce physical, genetic, and cytogenetic maps of the human genome and, ultimately, identify, sequence, and assign all 50,000 to 100,000 human genes (Collins). Genes successfully assigned to chromosomes will provide researchers with the necessary data to determine the specific location of the gene on the chromosome (Adams). Introduction In assigning a gene to a chromosome, tlie first step is to identify potential genes for assignment must be identified. A list of unassigned genes was obtained from the Genome Data Base and, from this list, the most recent additions to the list were selected (Fasman). Next, these identified genes were checked to see if they had previously been sequenced and, if so, whether there were at least 200 bases before or after the coding sequence (Caskey and Rossiter). Sections containing at least 200 bases before or after the coding sequence were used to select primer pairs by the Whitehead PRIMER Program (PRIMER). Primers selected for synthesis were synthesized using a Pharmacia DNA synthesizer. The synthesized primers were purified by molecular sieve chromatography and precipitated. The concentration of the primers was determined spectrophotometrically. A preliminary PCR of the unassigned gene was performed to determine whether a primer pair was successful at producing a detectable PCR product and to determine optimal PCR conditions. The preliminary PCR results were analyzed using gel electrophoresis and successful primer pairs completed PCR to produce a band in the gel. Only successful primer pairs were used in chromosome assignment. Chromosome assignment was made using a somatic cell hybrid mapping panel. Pools of chromosomes were constructed and PCR for the unassigned gene was performed with the pools. Results from chromosomal pool assignment were detected using gel electrophoresis. Once a pool was identified as containing the chromosome with the unassigned gene, then subsequent PCR reactions were performed to assign the gene to a chromosome in the pool. A gene was assigned to a specific chromsome if smiilar bands were detected for an individual chromosomal DNA PCR reaction and a human DNA PCR reaction. The data collected in this research project will be beneficial to the Human Genome Project because unassigned genes were assigned to chromosomes. Also, primer pairs that successfully assigned a gene to a chromosome could be used by other researchers working with specific chromosomes by providing them with the sequence of successful primer pairs. Successful primer pairs could then be used as sequence tagged sites (STSs) to construct a physical map (Davies). A cytogenetic map could also be constructed using yeast artificial chromosomes (YACs) identified by the primer pairs (Adams, Little ). Introduction to the Human Genome Project The Human Genome Project (HGP) is a world-wide, collaborative fifteen year research initiative developed by the National Institutes of Health (NIH), the Department of Energy (DOE), and the Human Genome Organization (HUGO)(Aldhous). In this project, scientists are working to identify all the genes of the human genome and to develop genetic and physical maps of the human genome and those of other model organisms. Development of these maps includes die determination of the location of the 50,000 to 1(X),000 human genes (Rose). Ultimately, this project seeks to sequence the entire genome of the human and other model organisms. The data collected in this project will allow for a better understanding of the structure and function of the human genome(CoUins). The NIH and DOE devised specific goals to be met by the Human Genome Project (Green). One of the goals calls for the improvement of mapping technology, in particular, mapping, sequencing, and gene identification. Another goal of the project is to produce genetic-linkage, physical, and cytogenetic maps of the human genome. A genetic-linkage map is a map indicating genetic distances between pairs of linked variable genes and a physical map is a representation of a group of recombinant clones that cover a chromosome from end to end (Rose). A cytogenetic map is the oldest type of map and it indicates the order and spacing of DNA markers along a chromsome relative to the chromosomal banding pattern (Caskey and Rossiter). This type of map is limited to a resolution of approximately ten milUon base pairs (Rose). Improvement in mapping technology will allow for the production of a more accurate physical map at a higher rate of efficiency (CoUins). The goal for the genetic map has already been exceeded and predictions of completing the HGP two years ahead of the scheduled 2005 completion date have been made (Beardsley). The information gathered by scientists must be easUy accessible to everyone because, as a world-wide collaborative effort, sharing data is critical in disseminating research results. Providing such a database has been a goal of die project and has led to the creation of the Genome Data Base (GDB) at Johns Hopkins University School of Medicme (Fasman). Additionally, the knowledge and understanding which result from the Human Genome Project raises important issues, such as how this information will be used. Thus, the final goal of the Human Genome Project is to address the ethical, legal, and social ramifications of identifying, mapping, and sequencing the entire human genome (Lippman). Mapping Genes Using PCR and Gel Electrophoresis In this research project, I worked with Dr. Robin Davies to map unassigned human genes to chromosomes. Genes for assignment were identified using the polymerase chain reaction (PCR) and subsequently analyzed using gel electrophoresis. PCR is a highly specific and sensitive technique in which defined DNA sequences are exponentially amplified through the action of a thermostable DNA polymerase enzyme (Faloona). The amplified product (a piece of DNA of a specific size) is detected via gel electrophoresis (Maniatis). In order to assign the gene to a chromosome, a somatic cell hybrid mapping panel is required (Zhang). A somatic cell hybrid mapping panel consists of a series of rodent- human cell hybrids, each of which contains a different human chromosome (Figure 1). The unassigned gene was assigned using PCR to examine the human/rodent somatic cell hybrid DNA and detecting the product of PCR with agarose gel electrophoresis to compare bands of the cell hybrids and the human control (Freshney, Zhang). Assignment of a gene to a chromosome was made when the human control PCR product matched the PCR product of one of the hybrids. Matching bands indicated the gene to be located in the human chromsome carried by the somadc cell hybrid (Table 1). The results of successful gene assignments can then be used to further the Human Genome Project by providing tools for the sub-chromosomal localization of genes. Polymerase Chain Reaction In order to assign a gene, a convenient method of gene identification must be available. Identification could be made by synthesizing the gene to be assigned, but it is impractical and too expensive to synthesize 200 to 1,000 bases of a gene. Another alternative would be to identify the gene using a Southern blot with hybrid DNAs to analyze a cDNA or genomic clone (Zhang). This method requires much more time and work because, in order for the gene to be detected, the hybrid DNA must be digested, electrophoresed, blotted, probed, washed, and detected. The cDNA or genomic clone must be labelled for use as a probe. On the other hand, PCR amplification followed by analysis with gel electrophoresis is faster and more straightforward (Zhang). A Southern blot would also require 5|ig of DNA, compared to only 5 - lOOng for PCR (Davies). Thus, more time, energy, and DNA is expended in gene identification through a Southern blot than in PCR. Gene assignment by PCR and gel electrophoresis is the most practical method of gene assignment because, in PCR, primers are used to locate the gene and then a DNA polymerase enzyme synthesizes DNA using the located gene as a template (Charlieu). Since PCR is an amplification of the located gene template, less DNA is used than in other techniques, and no additional steps, such as hybridization detection, are required (Faloona, Zhang). In PCR, a single-stranded DNA is used as a template for a thermostable DNA polymerase enzyme to elongate primers by complementary base pairing of deoxynucleotide triphosphates(dNTPs) in a buffered solution (MuUis). Primers locate and hybridize to a segment of the unassigned gene on the (single-stranded) DNA template. The DNA polymerase enzyme begins at the 3' hydroxyl group of the primer to catalyze the formation of phosphodiester bonds by "stringing" together "free" nucleotides (dNTPs) guided by complementary base pairing to the template strand, in the presence of a stable environment created by the buffering solution (Rolfs)(Figure 2). There are three steps to a typical PCR cycle: denaturation at 95°C, armeaUng at 48°C to 72°C, and extension at 72°C (Mullis). In denaturation, double stranded DNA is separated by breaking the hydrogen bonds connecting the two strands. The high temperature is needed to break the many hydrogen bonds. The separated single strands are each used as a template (Rolfs). Once denaturation is complete, primers are annealed to the single strand DNA template by decreasing the temperature to allow for hydrogen bonds to form between die complementary sites. The annealing temperamre is dependent on the lengdi and GC content of the primer; however, 50°C to 60°C is an acceptable range to begin annealing for a primer with approximately 50% GC content (Saiki, "Optimization"). The GC content refers to the percentage of G (guanine) and C (cytosine) and is important because the GC base pair has three hydrogen bonds versus the two hydrogen bonds connecting A (adenine) and T (thymine) and is, therefore, an indicator of DNA stability. Extension is the final step of PCR and refers to die complementary extension of the primer that has been annealed to the template DNA. This step must occur at the highest temperature possible because, by maximizing the extension temperature, incorrectly armealed primers are denatured more readily than correcUy annealed primers due to their unstable structure. The temperature of 72°C used in extension is close to die maximum activity temperamre of die DNA polymerase enzyme used in PCR (Saiki, "Primer"). The standard time used for extension is one minute for 1000 bases (Saiki, "Optimization"). For both the annealing and extension steps, nonspecific amplification products will result if the temperatures are held for too long. The DNA polymerase enzyme moves across the template strand 3' to 5' so that the synthesized product is made 5' to 3' (Innis). When these diree steps are repeated cyclically in PCR, die complementary strands completed in extension are denatured and separated to be used as templates in the next cycle. Cycling of the reaction makes it important that the melting temperature, die temperature at which half the double strands of DNA has denatured, of the two primers be similar because the extended primers need to separate from the DNA so that it can also be used as a template (Faloona). This means that new strands are synthesized exponentially because the denaturation provides two template strands and, after one cycle of PCR, double the amount of template strands are available. Primers A primer is an 1 8 to 24 base sequence that signals the beginning site of DNA synthesis to the DNA polymerase enzyme (Sharrocks). Primers are used to identify the gene to be amplified out of the 50,000 to 100,000 genes of the human genome because the many possible combinations of bases in a primer makes it highly specific to the region it will bind. A pair of primers is designed for each gene from the regions 5' or 3' to the coding sequence or from an intron. Primers cannot be selected from the coding sequence because this region codes for a working protein and, is therefore, conserved and the sequence may be seen elsewhere in the DNA (Voet). The primer locates the gene to be amplified by complementary base pairing to each strand of the double stranded DNA at the 3' hydroxyl end, which is the beginning of the region where DNA synthesis is initiated by the DNA polymerase enzyme (Figure 3). Possible primer pairs for PCR were selected using a computer software program called the Whitehead Primer Program Version 0.5 from the Massachusetts Institute of Technology (PRIMER). Primers selected from the possible primer pairs were synthesized using a computer-controlled Pharmacia DNA synthesizer. Sequences to be synthesized were entered into the computer and were synthesized by a method widely used, the phosphoramidite method (Figure 4)(Voet). There are two aspects of the chemistry involved in DNA synthesis: anhydrous and protection chemistry. Anhydrous chemistry is responsible for forming the phosphodiester bonds, which make up the backbone of DNA. In anhydrous chemistry, the presence of water in reagents and solvents decreases the coupUng efficiency. Coupling efficiency is the rate of success at which bases are "strung" together. Thus, care is taken to keep the solutions dry by carrying out all work quickly and including drying agents in most reagents. Groups not involved in a reaction are "blocked" by dimethoxytrityl (DMTr) in protection chemistry to insure correct order of reactions and coupling orientation and to maximize die integrity of the final product (Pharmacia). The first step of the phosphoramidite method of DNA synthesis is detritylation, in which the DMTr group of the nucleotide anchored to a solid support is cleaved to expose the 5' hydroxyl end by treatment with an acid, trichloroacetic acid (TCA). The solid support is an inert polymer encased in a plastic cassette on which DNA synthesis occurs, and the first nucleoside of the 3' end is bound to the support by a succinate bridge. The cleaved DMTr group is washed away and the amount washed away is detected and used to calculate the coupling efficiency. The 3' end of the nucleotide to be coupled to the growing chain is activated by tetrazole and is coupled to the 5' end of the growing chain by forming a phosphite triester bridge in a condensation reaction. Any remaining tetrazole, diisopropylamine, and unreacted nucleotides are washed away. Chains that failed to react are capped by acetylation to prevent the further synthesis of erroneous sequences with incoming nucleotides and to ensure that only the correct sequence is synthesized. Excess capping reagents are washed away. The phosphite triester bridge is oxidized with iodine in the presence of water to the phosphodiester form, thereby completing the coupling of one nucleotide to the growing chain. Once the sequence of interest is completely synthesized, it is cleaved from the solid support by treatment with ammonium hydroxide (Pharmacia). Template DNA In this project, PCR conditions were determined for human DNA and, then, the somatic cell hybrid mapping panel DNAs were used (Zhang). These human/rodent somatic cell hybrids are purchased from the Human Genetic Mutant Cell Repository and each contains a different human chromosome (Table 1). The DNA from these cells were grouped into pools that contained a specific number of human chromosomes. Each pool was subjected to PCR to look for a positive identification. A positive identification was determined when, after electrophoresis and staining with ethidium bromide, bands of the expected size were detected for a pool and matched the band detected for human DNA. The pool with the positive band was broken up into smaller pools containing fewer cell hybrids and a positive identification is again determined. This process was repeated until the gene was assigned to a somatic cell hybrid containing a specific chromosome (Catalog 1992/1993 and 1994/1995). Gel Electrophoresis The amplified unassigned gene sequence from PCR was analyzed using gel electrophoresis to determine chromosomal assignment. Gel electrophoresis is a technique that allows for determination of the size of DNA by the separation of molecules in a semi- soUd matrix using electric current to cause migration of the electrically charged DNA molecules. The pore size of the matrix is controlled by the number of crosslinks in an agarose gel, which is controlled by agarose concentration, and is used to separate the charged particles based on a desired size (Maniatis). Agarose gels are run on a horizontal slab and the positive electrode is placed furthest away from die weUs containing the negatively charged DNA. The DNA migrates through the gel towards the positive electrode, but the speed at which it can travel is Umited by its size. Following electrophoresis, the gels are stained using ethidium bromide. Ethidium bromide is used to stain the gel because it intercalates in the DNA and fluoresces under UV light (Figure 5). A single major band for each unassigned gene was detected using this method (Acquaah). Genes Assigned to Chromosomes Cartilage-derived Morpho genetic Protein 1 Cartilage-derived morphogenetic protein 1 (CDMPl-PEN) was assigned to the superfamily of transforming growth factor-6 (TGF-6) by using degenerate primer pairs from the highly conserved carboxyl-terminal region of bone morphogenetic proteins (BMP) in the articular cartilage of newborn calves. Bone morphogenetic proteins belong to the large TGF-6 superfamily, which are a large group of structurally related signaling proteins released as dimers and then cleaved to their active structure. Many of these proteins have been shown to be important at various stages of erabryogenesis and in adult animals. (Chang) Proteins produced by CDMP- 1 have been found at the stage of precartilaginous mesenchymal condensation and in the cartilaginous cores of developing long bones. Another protein known as cartilage-derived morphogenetic protein 2 has been identified and is 82% identical to the carboxyl-terminal TGF-B domain in CDMP-1. This similarity between CDMP-1 and CDMP-2 defines a new subfamily of proteins. Both genes produce proteins having bone-inductive activity and are preferentially expressed in cartilaginous tissues. Discovery of this novel subfamily is important because CDMP-1 and other BMPs have been demonstrated to be active in early skeletal development and CDMP-2 may be involved in later stages of skeletal formation. This means that these cartilage specific proteins can provide new information in regeneration of cartUage and in studying the cells responsible for production of these proteins (Chang). Asialo glycoprotein Receptor 2 Asialoglycoprotein receptor 2 (ASGR-2) is one of two receptor proteins, ASGR-1 and -2, which have a 58% protein homology. Two subspecies of ASGR-2 have been identified and they differ by a five-amino acid insertion in the COOH -terminal extracytoplasmic domain (Spiess). Asialoglycoprotein receptors have been found on mammahan parenchymal hepatocytes and bind desialylated glycoproteins. Glycoproteins are proteins with a covalently bound carbohydrate group and are found in most forms of living things. The functions of glycoproteins are highly varied, including those of enzymes, transport proteins, receptors, hormones, and structural proteins. The asialoglycoprotein receptors bind desialylated glycoproteins and the receptor-ligand complex is taken into the cell and transported to an acidic sorting compartment. The receptor is then recycled back to the surface of the cell and the Ugand is degraded by the lysosomes. This activity has been 10 observed in both mice and humans. Comparison of the human-1 (H-1) and human-2 (H-2) to rat-1 (R-1) and rat-2 (R-2) have shown more homology to exist between H-1 and R-1 and H-2 and R-2 than to each other, suggesting that these genes evolved before the evolutionary separation of humans and rats (Spiess). 11 Materials and Methods Identifying Possible Unassigned Genes for Mapping The Genome Data Base (Fasman) was accessed using Netscape, a world wide web browser, to obtain a current list of unassigned genes. The assignment status of randomly selected new entries on the list was verified by searching for current information on the GDB. Genes selected for assignment had previously been sequenced and contained a minimum of two hundred bases in at least one of their untranslated regions (5' untranslated, 3' untranslated, or introns). An untranslated region of appropriate length (at least 200 bases) was excerpted from each sequence and was placed in a separate computer file for primer selection. Primer Selection Primer pairs for each unassigned gene were selected using computer files of the untranslated sequence from the GDB and a primer selection program called the Whitehead Primer Program from the Massachusetts Institute of Technology (PRIMER). The program was used to deermine: which part of the sequence should be amplified; whether possible primers would be compatible to the conditions of the PCR (e.g. annealing temperature); if the primers have a significant degree of complementarity to repeat sequences; whether the primer pairs have a significant degree of complementarity to each other; and whether the PCR product would be of the desired size and GC content. If any repetitive sequences were found, the program was designed to identify them and avoid producing primers in the repetitive sequence, to ensure unique PCR products for each gene. The program produced three possible primer pairs differing in product size range: 100 - 150bp, 150 - 250bp, and 250 - 4(X)bp. Primer pairs selected for synthesis had an even distribution of bases; similar melting temperatures; a 45 - 55% GC content; and mononucleotide chains of less than four bases. Primer Synthesis Primers were synthesized using an automated DNA synthesizer, the Gene 12 Assembler Special/4 Primers. The synthesizer and all reagents for synthesis (Appendix I), were obtained from Pharmacia Biotech. Before synthesis, the argon level of the instrument was checked. The instrument was maintained under pressure with argon to prevent the evaporation and water contamination of the reagents. The pump hose was connected to the instrument. The instrument was purged of reagents remaining in the tubing and a solid support cartridge corresponding to the 3' base of the sequence to be synthesized was placed in the first column. (Unlike DNA synthesis in the cell, automated DNA synthesis proceeds in the 3' to 5' direction.) Next, the pump was calibrated and the new calibration value was only accepted if the new caUbration value had a difference of less than 5% from the old caUbration value. A proper cahbration value is important because the calibration determines when the monitor will measure the detritylation value, which is used to calculate coupling efficiency. Therefore, if the caUbration is not correctly adjusted, then the instrument will not calculate the coupling efficiency correctly. Once the instrument was prepared for synthesis the sequence of the primer to be synthesized was entered, edited, described, and saved. Synthesis was initiated and then monitored to insure efficiency. Cleaving Primer from the Solid Support When synthesis was completed, the cartridge was removed from the column and placed in a sterile 1.5mL Eppendorf tube. The cartridge was centrifuged at 2500rpm for 1.5 minutes in an Eppendorf centrifuge to remove any remaining acetonitrile. Following centrifugation, the cartridge was transferred to a sterile 1.5mL Eppendorf tube using a sterile 22 gauge / 1.5 inch Becton-Dickinson syringe needle. The tube was tapped to secure the cartridge in the tube. The cartridge was covered with ImL ammonium hydroxide, sealed with parafilm, and placed in a jar containing ammonium hydroxide overnight (at least eight hours) at room temperature to cleave the synthesized DNA from the solid support. Primer Purification The tube containing the cartridge was removed and the cartridge was transferred to 13 a second sterile 1.5mL Eppendorf tube, saving the ammonium hydroxide remaining in the tube. The cartridge was centrifuged in an Eppendorf centrifuge at 2500rpm for 1.5 minutes to remove ammonium hydroxide. The ammonium hydroxide centrifuged out of the cartridge was combined with the previous ammonium hydroxide saved and the volume was adjusted to l.OmL using sterile double distilled water. Primers were purified by molecular sieve chromatography using Pharmacia Biotech NAP- 10 columns to remove capped sequences. The top and bottom cap of the column were removed and solution drained off. The column was supported in a vertical position and the gel bed was washed three times with 5mL sterile double distilled water. Once the water had completely entered the gel bed, the sample was poured over the gel bed. The purified sample was eluted with 1.5mL sterile double distilled water and collected into a sterile 1.5mL Eppendorf tube. Ethanol Precipitation of Primers The purified sample was aliquoted at a maximum volume of 400)il in sterile 1.5mL Eppendorf tubes. For every 400 |iL of purified primer, 40 [iL 3M sodium acetate, 4 |iL IM MgCl2, and 935 p.L 95% EtOH were added and mixed by inverting the tubes. The tubes were stored for a minimum of two hours at -80°C in a Revco deep freezer to concentrate and purify the primers via precipitation. Isolation of Primers The tubes were removed from the -80°C freezer and centrifuged for ten minutes at 14000 rpm in an Eppendorf centrifuge. Following centrifugation, the supernatant was removed and the pellets were rinsed with l.OmL 70% EtOH in lOmMTris ImM EDTA, pH 8.0 (TE) and re-centrifuged as above. The supernatant was removed and the pellets were dried in a 600mmHg vacuum and dissolved in 40 |iL TE. The concentrated dissolved primers were collected into sterile 1.5mL Eppendorf tubes. The tubes that had contained the pellets were washed with 40 |iL TE by transferring from tubes. The TE used for washing was collected into the tube with the concentrated dissolved primer to maximize 14 yield and to prevent loss of oligonucleotides clinging to the tubes. Determining the Concentration of Concentrated Primer The concentration of primer stock solutions was determined by measuring the absorbance (A) of the sample using a Beckman DU-600 spectrophotometer at 260nm. The sample was measured using ImL of a 1/250 dilution of the concentrated stock. The concentration of the stock solution was determined using the measured absorbance in the following equation (Maniatis): 1 A = 33rag/mL Measured Absorbance X 33mg/mL = Concentration of Dilution Concentration of Dilution X 250 dilution factor = Stock Concentration PCR of Unassigned Genes Genes were analyzed using PCR, performed in a Power Block I System from Ericomp. Master mixes of the primer pair were made in a sterile Eppendorf tube and contained sterile double distilled water; Gibco lOx buffer +Mg''"''" (1.5mM); Gibco dNTPs (lOmM); lOOng/mL of each primer, and Gibco Taq (5U/|iL)(Appendix 11). The master mix (19|iL) was pipeted into a thin wall PCR tube from United Laboratory Plastics. One microliter of lOOng/mL dilution of Sigma (human placental), AHH-1 (human lymphoblast), or Gibco (K562 human transfer cell Une) DNA was pipeted into each PCR tube. The positive control used in each reaction contained Gibco DNA and Gibco primers for brain-derived neurotrophic factor. The tubes were set up for PCR and placed in the thermal cycler and cycled x 30. The program for each reaction was the same except for the annealing temperature. An initial denaturation at 94°C for four minutes was followed by 30 cycles of denaturation at 94°C for 30 seconds; annealing at approximately two degrees below the melting temperature of the primers for 20 seconds; and extension at 72°C for 10 seconds. Final extension was for five minutes at 72°C. Following final extension, the tubes were either held at 4°C for 15 minutes or placed in the freezer until the PCR products were examined 15 by gel electrophoresis. The stringency of the reaction was adjusted by varying the concentration of Mg+'*' and/or the annealing temperature. Preparation of Gels for Gel Electrophoresis All gels used were 1.5% agarose with 5 |iL ethidium bromide incorporated into the gel. In a 250mL flask, 1.5g Sigma agarose was combined with 80mL double distilled water and microwaved. Next, 20 mL 5x TBE and 5 |lL lOmg/ml ethidium bromide were added. Examination of PCR Product Using Gel Electrophoresis The gel was placed in the electrophoresis chamber and approximately 1.8L Ix TBE (lOmM tris, pH = 8; lOmM borate; lOmM EDTA) was added. Each 20 |iL PCR reaction was augmented with 4 |iL of 2.5% bromophenol blue sample buffer, and 22 |iL of the PCR product was pipeted into the well. The first, second, and twelfth wells contained DNA markers: bacteriophage lambda DNA digested with Hin D HI; bacteriophage PhiX 174 DNA digested with Hae III; or 1Kb ladder (Appendix III). Gels were run at 180 volts using a Bio-Rad Model 1000/500 Power Supply for thirty minutes and viewed using a Fotodyne Foto/Prep I UV hght source. Pictures of the gels were taken with a Fotodyne Polaroid camera and type 667 black-and-white film. Chromosome Assignment of Gene Using PCR Twelve pools containing two different chromosomal DNAs from a mapping panel obtained from the NIGMS Human Genetic Mutant Cell Repository were designed for chromosomal assignment. A master mix same as "PCR of Unassigned Genes" was made in a sterile Eppendorf tube. The master mix was mixed by inverting the tube, and 19 |J.L of the mix was pipeted into each PCR mbe. One microUter of the template DNA from one of the twelve pools was added to each tube so that each reaction tube contained DNA from two chromosomes. The positive control contained human DNA (from all chromsomes) and the negative controls were mouse and Chinese hamster DNA from the same mapping panel. 16 The reaction tubes were placed in the Power Block I System thermal cycler and run through 30 cycles of PCR, according to the same program used to analyze the primers earlier. The PCR products were examined using gel electrophoresis as previously described. Observed bands seen in the gel were examined to determine which pool the gene was observed. PCR was repeated to determine the specific chromosomal location of the gene using separate reaction tubes for each of the two chromosomes present in the positive pool. 17 Results Primer Selection and Synthesis The search of the GDB produced a Ust of 1 697 unassigned genes, and 62 recently submitted genes were selected for potential mapping. Thirty-one of these genes were further selected for mapping because they had previously been sequenced and had a minimum of 200 bases before or after their coding sequence. Nineteen genes were examined using the primer program and twenty-nine primer sequences were selected for synthesis. Twenty-six syntheses were attempted and 16 syntheses were successful (Tables 2 and 3). The success of a synthesis was determined by whether or not the sequence to be synthesized was successfully coupled (Table 4). Eleven of the primers synthesized had coupling efficiency percentages above 90% and most produced a high (above 1000 [ig/mL) primer stock concentration; however, the forward primer for TINUR, which had a 100% coupling efficiency, produced the lowest primer stock concentration at 0 |ig/mL and the reverse primer for CATR also had a low stock concentration despite its high coupling efficiency percentage. This discrepancy between coupling efficiency percentage and primer stock concentration prevented a direct relationship from being constructed. The three primers that produced low (below 1000|J.g/mL) primer stock concentrations had below 90% coupUng efficiency. Primers not successfully synthesized were automatically aborted during synthesis. If the cause was a loss of reagents, than synthesis was aborted towards the end of synthesis, with approximately 1 - 3 bases remaining (Table 5). Inspection of Table 5 indicates that for these events the coupling efficiency data was similar to a successful synthesis (Table 6) until the last base in which the Detrit Start and Detrit Value were both zero due to the loss of synthesis solvent. Miscalculation of the amount of amidites, tetrazole, dichloroethane, acetylnitrile, or detritylation solution was due to a discrepancy between the computer monitoring of solvents used and the acmal amount available. A number of syntheses were aborted due to detritylation problems. Any syntheses that were 18 not completed due to low detritylation values were stopped after approximately the third base coupled in an effort to conserve solvents. Synthesis continued despite low detritylation values would waste solvents, since, if no detritylation is occuring, then bases will not be coupled. The detritylation values in this case would drop dramatically after the first coupled base and continue to decrease (Table 7). Synthesis was once aborted due to a break in the pump hose causing a leak. No coupling efficiency data was available because no couphng was made prior to the pump hose break. Pump hose tears were not predictable and, therefore, inspection of the detritylation values was not effective in determining the reason for a stopped synthesis until the pump hose was inspected. Concentration Determination of Synthesized Primers Concentration of primers measured at a 250x dilution varied from a high of 4799.0|ig/mL (reverse primer for RMSAl-PEN) to a low of 0|j,g/ml (forward primer for TINUR-LSB). A direct relationship between the average couphng efficiency and the primer stock concentration calculated from the absorbance could not be determined (Table 4). Preliminary PCR In order to estabhsh which primer pairs worked and to determine the optimal conditions for PCR, preliminary trials were performed for all primer pairs. The first preliminary PCR was performed for CDMP 1-1 (329bp) and CDMPl-2(205bp), which were prepared from the 5' and the 3' untranslated regions, respectively, of the cartilage -derived morphogenic protein 1 cDNA sequence (Chang)(Figure 6). WeUs 1 and 12 contained lambda Hin D III and well 2 contained PhiX Hae EI marker DNAs. Wells 4 through 6 contained human DNA with CDMPl-1 primers and no bands were visible indicating the CDMPl-1 primer pair not to be an assignment candidate. WeUs 8 through 10 contained human DNA with the CDMPl-2 primer pair. Bands were visible in all three human DNA PCR reactions and stronger bands were visible for the PCR performed with Gibco and Sigma DNA than with the AHH- 1 DNA. The bands visible were 205 bp in 19 size, which was in agreement with the expected value. The band presence indicated the CDMPl-2 primer pair to be a candidate for assignment. The second gene sequence to be examined was the asialoglycoprotein receptor 2 (ASGR2) cDNA sequence from the 3' untranslated region (ASGR2-2)(Spiess). The first preliminary PCR for this gene was performed using the primer pair ASGR2-2 with a 58°C aimealing temperature (Figure 7). Wells 1 and 7 contained lambda Hin D in and well 2 contained PhiX Hae in marker DNAs. Wells 4 through 6 contained ASGR2-2 with human DNA. The expected PCR product size was 21 Ibp, no band was detected, rendering ASGR2-2 unusable in assignment. The next preUminary PCR for this gene was performed using the primer pair ASGR2-1 (Figure 8). Wells 1 and 12 contained lambda Hin D ni DNA and well 2 contained PhiX Hae III marker DNAs. Wells 4 through 6 contained ASGR2-1 with human DNA. Faint bands of 146bp were only visible in wells 4 and 5, which were Gibco and Sigma human DNA. The faintness of the band indicated the PCR to be too stringent. A second preUminary PCR for ASGR2- 1 was performed and the stringency of the reaction was reduced by decreasing the annealing temperature to 57 °C (Figure 9). Wells 1 and 10 contained lambda Hin D in DNA and well 2 contained PhiX Hae HI marker DNAs. WeUs 3 through 5 contained ASGR2-1 with human DNA. PCR with Gibco DNA in well 3 did not produce a distinct band, but a slight smear instead. The strongest band was produced in well 4 which contained the PCR with Sigma DNA. The weakest band was produced in the PCR using the human DNA from the mapping panel. The gel identified ASGR2- 1 as the primer pair to be used in chromosomal assignment of this gene and also demonstrated that annealing at 57°C produced stronger signals than annealing at 58°C. Annealing at 57°C also increased the amount of background DNA present at lower molecular weights, which can be identified by the faint bands of smaller size in Figure 9. The third gene sequence to be examined was the regulator of mitotic spindle assembly 1 (RMS A). The primer pairs were prepared from the 3' untranslated region of 20 the RMSA cDNA (Margalit, Yeo). The first preliminary for RMSA was performed at an annealing temperature of 57.5°C (Figure 9). Well 1 contained lambda Hin D HI DNA and well 2 contained PhiX marker DNAs. Wells 4 through 6 contained RMSA with human DNA. A very faint 249bp band was visible in well 4 which contained Sigma DNA. Chromosomal Assignment Cartilage-derived morphogenic protein 1 Initially, chromosome assignment of CDMPl was attempted using six different pools of chromosomal DNA (Table 8). Each pool contained DNA from 4 - 5 different chromosomes. Assignment using these pools was not successful because no band was detected for any of the pools (Figure 10). The positive PCR control and the positive control using human DNA each produced a band, indicating the PCR to have been a successful reaction. No bands were detected in the mouse and Chinese hamster negative controls. Since no bands were detected, the pool sizes were decreased and the number of pools were increased in order to increase the concentration of the chromosomal DNA in the cell. Chromosome assignment for CDMPl was next attempted using 12 different pools of chromosomal DNA, each pool containing DNA from only two different chromosomes (Table 9). A faint band was detected in the PCR product of pool 2, a pool which contained DNA from chromosomes 20 and 21 (Figure 1 1). PCR was performed for chromosomes 20 and 21 and a band was detected for chromosome 20 (Figure 12). This final result determined the location of CDMPl to be on the 20th chromosome. Chromosome assignment for CDMPl was repeated for verification of results using chromosomal DNA from chromsomes 2 and 20. A band was detected for chromosome 20 and no hand was detected for chromosome 2 (Figure 13). Asialo glycoprotein receptor 2 Chromosome assignment of ASGR2 using 12 different pools of chromosomal DNA at 57°C with a l.SOmM Mg++ concentration produced faint bands (Figure 14). 21 Wells 1 and 2 contained 1Kb ladder marker DNA and well 12 contained lambda Hin D m DNA. Faint bands were detected in wells 6, 7, and 8, which contained pools 4, 5, and 6. According to this gel, the gene was located on chromosome 17, 16, 15, 14, 13, or 12. Assignment of the gene to a chromosome was attempted using chromosomal DNA from 17, 16, 15, 14, 13, and 12 at 1.50mM Mg++, but no bands were detected and the reactions were therefore unsuccessful (Figure 15). Chromosome assignment was again attempted at a decreased stringency by increasing the concentration of Mg++. Parallel reactions were run at 1.75mM and 2.00mM Mg"*""*" and the PCR products were run in a longer gel to allow for more separation between the PCR product bands and the background DNA. Assignment at an increased Mg"^"*" concentration was unsuccessful because no bands were observed (Figures 16 and 17). 22 Discussion Chromosome Assignment Cartilage-derived morphogenetic protein 1 The cartilage-derived morphogenetic protein 1 gene was determined to be on chromosome 20. The band for chromosome 20 is faint and none was present for chromsome 21. The assignment was repeated to demonstrate that the assignment was reproducible. Although the bands observed for this gene were faint, they were detected in the gel and indicated that, using the primers for the gene, PCR did occur. The band signal for CDMPl could be increased by further decreasing the stringency of the reaction using a decreased Mg++ concentration. The primer pairs developed for CDMPl would be useful in the assignment of CDMPl to the physical map of chromosome 20. Further testing of this gene should include cytogenetic mapping; ideally, with a YAC containing this gene. Cytogenetic mapping would be performed using fluorescence in situ hybridization and should give a signal on the appropriate chromosome (Caskey and Rossiter). Determination of the chromosome assignment of this gene would be significant because it is a new subfamily of proteins. Chromosomal determination of CDMP2 would now be interesting to determine if this subfamily is inherited together. In the future, this subfamily could be used in gene therapy to treat arthritis. Asialoglycoprotein receptor 2 The gene for asialoglycoprotein receptor 2 was determined to be located on either chromosome 17, 16, 15, 14, 13, or 12. A specific chromosome assignment was attempted at three different Mg++ concentrations, but was unsuccessful. Further testing of this primer pair is necessary to better optimize the PCR conditions and continue chromosome assignment. 23 Preliminary PCR Asialoglycoprotein Receptor 2 A primer pair for PCR of asialoglycoprotein receptor 2 was successfully identified. The signal produced by the band was weak, decreasing the stringency of the reaction by decreasing the anneahng temperature produced a stronger band. As the annealing temperature was decreased, the signal observed was stronger, but more background bands were also detected. Therefore, an annealing temperature of 57°C was selected because PCR at this temperature produced a stronger signal with as httle background as possible. Although the primer pair has been shown to work, further testing is necessary to optimize PCR conditions. Determination of a successful primer pair for ASGR2 is significant because it will allow for the gene to be assigned to a chromosome using the primer pair as a sequence tag site to determine the sub-chromosomal location. Regulator of mitotic assembly 1 A primer pair for the regulator of mitotic assembly 1 (RMSA) gene was determined. Optimal conditions for PCR of this gene must still determined. The PCR reaction for this gene may be optimized by adjusting the annealing temperature and the concentration of Mg"*""*". Once conditions for PCR have been determined, the chromosomal location of this gene could be determined. The primer pairs could then be used as an STS to determine the sub-chromosomal location of the gene. Trouble Shooting Primer Synthesis The main problem encountered in primer synthesis was the lack of detritylation. Problems with detritylation were first encountered when a new botde of acetonitrile was added to the instrument. Detritylation problems are typically indicative of a water contamination of the system and/or solvents. The instrument was flushed on three separate occasions by back pumping acetonitrile through the system. Since these problems began after the new botde of acetonitrile was connected to the 24 instrument, it is probable that the acetonitrile may not have been properly dried. If this was the case, then the water contamination caused by the wet acetonitrile may have been made worse each time new amidites were dissolved and connected to the instrument because they were dissolved in the same wet acetonitrile. Therefore, flushing the instrument may have perpetuated the problem by spreading wet acetonitrile. In the future, solvents that have molecular sieves added to them may be dried more thoroughly by taking better precautions. Prior to adding the sieves, the sieves could be heated to approximately 60°C to insure their dry state. After adding the heated sieves, the botde connections on the instrument should be checked to assure that no debris is found in the seaUng ring; this should prevent any leaks. The solvents should be connected to the instrument and the pressurized argon should be allowed to flow into the bottles to displace the air introduced by cormecting the bottles. The new solvents should be left over night on the instrument and, just before initiating synthesis, the pressurized argon should be allowed to flow into the solvents again. In the future, if detritylation is not happening in DNA synthesis, then a different trouble-shooting approach would be more efficient in the use of time and money. A new botde of acetonitrile should be prepared as indicated above. New amidites should be prepared using the new bottle of acetonitrile. This acetonitrile should aso be used to flush the instrument to remove any remaining water. Concentration Determinadon of Synthesized Primers Problems were encountered in determining the absorbance of primers using the Beckman DU-600 spectrophotometer. The spectrophotometer was producing absorbance readings that varied each time a primer absorbance was measured so that data could not be repUcated. Next, the spectrophotometer began reading negative absorbance readings; meaning, the TE blank was absorbing more than the primer samples. In order to verify the working order of the spectrophotometer, a series of dilutions of a known concentration of sheared salmon sperm DNA was used. The purpose of this 25 assay was to produce absorbance readings diat decreased as the concentration decreased if there were no problems with the instrument. Some variation was seen, but, overall, a relative decrease was seen. The spectrophotometer in the chemistry department was used to confirm the absorbance readings of the primers measured by the biology spectrophotometer. Since no problem with the instrument was ascertained, it is likely that the discrepancy in concentration readings was due to incorrect storage of the primers. The primers were being stored in the refigerator at 4°C, but should have been stored in the freezer at 20°C. Degradation may have occurred as a result. Automated Thermal Cycler In the first PCRs performed using the automated thermal cycler, the instrument failed to begin final extension at 72°C for 4 minutes. The technician for the company was contacted and he suggested changing the final extension temperature to 71.9°C because the last step prior to final extension was also at 72°C; therefore, the computer may have had a problem with distinguishing between the two steps due to their same temperatures. The "overshoot low" control on the thermal cycler was also lowered to permanendy correct the problem. 26 Conclusion The two human genes, cartilage-derived morphogenic protein 1 and asialoglycoprotein receptor 2, were assigned to chromosomes. CDMPl was assigned to chromosome 20 and ASGR2 was located to three different chromosomal pools using physical mapping techniques. Cytogenic mapping using fluorescence in sitii hybridization should be performed for CDMPl to determine the chromosomal band location of the gene (Caskey and Rossiter). Further testing of the Mg"*""^ concentration and anneahng temperature of ASGR2 is necessary to optimize the PCR conditions in order to determine the exact chromosome location of the gene. Once located to a chromosome, the primer pair may be used by others to determine its specific location along the chromosome (Davies). A possible primer pair for regulator of mitotic spindle assembly 1 was determined. Further testing of RMS A is required to optimize its PCR conditions and to assign the gene to a chromosome. Researchers working on the Human Genome Project may use the primer pairs and chromosome assignments determined to further their work. The primer pairs and chromosome assignments would provide others with the information necessary to perform further mapping of the three genes evaluated here. 27 Table 1 - Somatic Cell Hybrid Mapping Panel from NIGMS Human Genetic Mutant Cell Repository by fusing human and rodent cells. In cell lines such as GM/NA10791 and GM/NA10888, the chromosome is present in all of the cells.Not all genes are present at 100%, for example in the cell line GM/NAl 1418, only 88% of the cells contain chromosome 14. A percentage of cells in lines GM/NA07299 and GM/NA10478 both contain two different chromosome. (Catalog, 1992/1993) NIGMS HLWAN/RODENT SOMATIC CELL HYBRID MAPPING PANEL #2 PERCENTAGE OF CELLS WITH HUMAN CHROMOSOMES CHROMOSOME* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 HYBRID/DNA GM/NA07299 33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 85 0 GM/NAl 08268 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10253 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10115 0 0 0 97 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10114 0 0 0 0 93 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10629 0 0 0 0 0 98 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10791 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10156B 0 0 0 0 0 0 0 96 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NAl 0611 0 0 0 0 0 0 0 0 69 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10926B 0 0 0 0 0 0 0 0 0 76 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10927A 0 0 0 0 0 0 0 0 0 0 92 0 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10868 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 GM/NA10898 0 0 0 0 0 0 0 0 0 0 0 0 92 0 0 0 0 0 0 0 0 0 0 0 GM/NA10479 0 0 0 0 0 0 0 0 0 0 0 0 0 88 0 0 0 0 0 0 0 0 0 0 GM/NA11418 0 0 0 0 0 0 0 0 0 0 0 0 0 0 88 0 0 0 0 0 0 0 0 0 GM/NA10567 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 98 0 0 0 0 0 0 0 0 GM/NA10498 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 96 "0 0 0 0 0 0 0 GM/NAllOlO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 72 0 0 0 0 0 0 GM/NAl 0449 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 96 0 0 0 0 0 GM/NA10478 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 76 0 0 0 0 GM/NA10323 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 86 0 0 0 GM/NAl 0888 0 0 0 0 r 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 GM/NA06318B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 GM/NA06317 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 96 28 Table 2 - Name of the genes primers were synthesized for; gene symbol; and PCR designation. Name of Gene Gene Symbol PCR Designation cartilage-derived morphogenic protein 1 CDMPl-PEN CDMPI-1 CDMPl-2 asialoglycoprotein receptor 2 ASGR2 ASGR2-1 ASGR2-2 tumorigenic conversion associated gene CATRl-LSB CATRl-LSB transcriptionally inducible nuclear receptor TINUR-LSB TINUR-LSB regulator of mitotic spindle assembly 1 RMSAl-PEN RMSAl-PEN hepatoma-derived growth factor (heparin-binding) HDGF-LSB HDGF-LSB cytochrome c oxidase subunit Via polypeptide 1 C0X6A1 C0X6A1 Table 3 - PCR designation, sequence, primer type, 5' / 3' to coding sequence, and product length of primers synthesized. Annealing temperature of successful primer pairs. Gene Symbol Sequence 5' to 3' Forward / Reverse Primer 5' / 3' to Coding Sequence Product Length (bp) Annealing Temp. (Celsius) CDMPl-1 TGGCAGGAGC ATCTACACAG forward 3' 329 N/A GGAAGTCACC AGGCACAAAT reverse CDMPl-2 TGCACGTCTG GATACGAGAG forward 5' 205 58 TGACACCAAA GAGAACAGCG reverse ASGR2 TTCCTACGCC ATTGAAGAGG reverse 3' ASGR2-I CACTGAGACA GGGGTATGGG forward 146 57 ASGR2-2 TAACCCATAC CCCACACCTG forward 211 N/A CATRl-LSB CTCTAGGTAT CAGTGGGGCG reverse 3' 216 N/A TTTCCAAGGG CCAATCTATG forward TINUR-LSB CTGAACTGCA ACAACCAAGC reverse 3' 303 N/A CAGAGATAGC CGTGTGAGCA forward RMSAl-PEN GTTGTAGCAG GAAAGCAGCC forward 3' 249 57.5 TACCCCCACC CAAAGTCATA reverse HDGF-LSB TCATCAAGAG AATTTGGGGC forward 3' 313 N/A COX6A1 TTTAAGCCAT CTCCTGCCAC reverse 3' 329 N/A 29 Table 4 - Coupling efficiency and concentration of primers synthesized. PCR Designation Forward / Reverse Primer Coupling Effiency(%) Primer Stock Conc(|ig/mL) CDMPl-1 forward 100 2251.4 reverse 100 2856.9 CDMPl-2 forward 99.1 2932.9 reverse 93.6 4131.6 ASGR2 reverse 100 4115.1 ASGR2-1 forward 100 1360.4 ASGR2-2 forward 86.4 614.60 CATR-LSB forward 72.0 154.60 reverse 94.4 830.78 TINUR-LSB forward 100 0 reverse 65.8 12.458 RMSAl-PEN forward 100 4490.3 reverse 99.7 4799.0 HDGFl-LSB forward 100 4079.0 COX6A1 reverse 76.8 42.157 Table 5 - coupling efficiency data from DNA synthesis interrupted by loss of solvents. Position Base Detrit Start Detrit Value 20 C 0.44 81 19 A 0.44 211 18 C 0.44 187 17 C 0.45 175 16 G 0.42 26 15 T 0.45 156 14 C 0.45 144 13 C 0.45 135 12 T 0.46 172 11 C 0.45 135 10 T 0.46 142 9 A 0.45 141 8 C 0.46 126 7 c 0.46 128 6 G 0.42 28 5 A 0.46 124 4 A 0.45 135 3 T 0.46 117 2 T 0.46 1 1 T 0.00 0 30 Table 6 - Coupling efficiency data from DNA synthesis not detritylating. Position Base Detrit Start Detrit Value 20 A 0.44 65 19 A 0.41 11 18 C 0.43 4 17 A 0.43 6 16 C 0.43 3 Table 7 - Typical coupling efficiency data for successful DNA synthesis. Position Base Detrit Start Detrit Value 20 C 0.49 88 19 C 0.48 143 18 G 0.46 121 17 A 0.48 151 16 C 0.49 152 15 G 0.48 131 14 A 0.49 163 13 A 0.51 162 12 A 0.51 170 11 G 0.50 137 10 G 0.50 137 9 A 0.52 160 8 C 0.54 157 7 G 0.53 135 6 A 0.55 164 5 T 0.58 165 4 G 0.54 139 3 T 0.57 164 2 T 0.57 166 1 G 0.55 140 31 Table 8 - Initial 6 pools used in chromosome Pool Chromosome Initial Cone (ng/^tl) 1 5 371ng/|il 2 368 22 376 13 377 17 370 2 Y 358 X 366 12 364 6 355 11 357 3 4 343 1 352 19 326 7 327 4 8 311 21 314 20 313 10 323 5 3 310 18 283 15 285 14 292 6 16 330 9 351 assignment. 32 Table 9-12 pools used in chromosome assignment. Pool Chromosome Initial Cone 1 X 366ng/|il 22 376 2 21 314 20 313 3 19 326 18 283 4 17 370 16 330 5 15 285 14 292 6 13 377 12 364 7 11 357 10 323 8 9 351 8 311 9 7 327 6 355 10 5 371 4 343 11 3 310 2 368 12 Y 358 1 352 33 Fi2. 1 - Human rodent somatic cell hvbrid showing chromosomes 34 Fig. 2 - Diagram of the polymerase chain reaction(Cooper) Reaction mixture includes DNA sample: two single-stranded primers, eacti with a 20-base sequence complementary to the 3' end of one strand of the target sequence; heat stable Taq polymerase; and deoxyribo- nucleoside tnphosphates (dNTPs). Phase 1 Denature unamplified DNA at 95'C to form single-stranded templates. Phase 2 Anneal pnmers to template at about 60°C. Phase 3 Synthesize new strands at 72°C. dGTP <^ ^irnerR ^°TP ''^"o'ym, Target sequence e^ase CYCLE 1 i 5' ^^ — 3' 3' ^— ^5' 1 X ' I Unamplified DNA (Hyarogen Donds berween the two ONA strands are shown as vertical lines i Template strands Each pnmer binds to its complementary sequence at the 3' end of a strand of the target sequence. Each new strand is synthesized in the 5'-to-3' direction. Phases 1 and 2 Denature products of Cycle 1 and anneal pnmers to template strands. Phase 3 Synthesize new strands. Phases 1 and 2 Denature products of Cycle 2 and anneal primers to template strands. Phase 3 Synthesize new strands. CYCLE 2 - First two template strands that _ span the target sequence only CYCLE 3 ■ Eight template strands that span the target sequence only Continue for 20 to 30 cycles to produce over 10^ copies of target sequence. 35 Fig. 3 - Primers bound at the 3' hydroxyl end of the template strand. (Rolfs) ^^-•^ ^ oncinal template f-t-l _ 3' primary products original template (-; Fig. 4 - Phosphoramidite method of DNA synthesis(Pharmacia) Detntylaiion W DMTr .A \; A DMTr A G = = Monomer bases • Ease protectirg group DMTr DMTr protecting group Phosphite protecting group (=3-cyance:hyn O Diisopropylamine group ^ Tetrazole - Cacping Group Wash W Activation 0 P B OMTr . \' A ► P E DMTr Coupling Wash Capping W ' '■.■■; A P .DMTr /\\-\-\\\^Ai \ •. \ '..\. A P jMTr /W.WAMA' Wash Oxidation \. ^. \ \ \.i_A_ P • 0 • .DMTr W \AV\\\iA, Wash Fig. 5 - Ethidium ion intercalates with DNA. H.N. NH2 37 Fig. 6 - Preliminary assignment for CDMPl-1 (329bp) and CDMPl-2 (205bpi. CDMPl-1 was determined unusable and CDMPl-2 was shown to be a productive primer pair. Faint band detected in well 4 for pool 2 containing chromosomes 20 and 2 1 . Lanes 1 and 12 = lambda Hin D III DN'A. Lane 2 = PhiX Hue III DNA. Lane 3 = TE and CD.MPl- 1 . Lane 4 = Gibco DNA and CDMPl-1. Lane 5 = Sisma DNA and CDMPl-1. Lane 6 = AHHl DNA and CDMPl-l. Lane ~ = TE and CDMPl-2. Lane 8 = Gibco DNA and CDMPl-2. Lane 9 = Sigma DNA and CDMPl-2. Lane 10 = AHHI DNA and CD.MPl -2. Lane 11 = Gibco Primer mi.x and DN.A. 38 Fig. 7 - Preliminary' PCR for ASGR2-2 (21 Ibp). No bands detected determinining ASGR2-2 to be an unusable primer pair. Lane 1 and 7 = lambda Hin D III DNA. Lane 2 = PhiX Hue III DNA. Lane 3 = TE and ASGR2-2. Lane 4 = Gibco DNA and ASGR2-2. Lane 5 = Sisma DNA and ASGR2-2. Lane 6 = AHHl and ASGR2-2. 39 Fig. 8 - Preliminary PCR of ASGR2-1 ( 146bp) indicating it to be a successful primer pair. Famt band detected in lanes 4 and 5. Lanes 1 and 12 = lambda Hin D III DNA. Lane 2 = PhiX Hae III DNA. Lane 3 = TE and ASGR2-1. Lane 4 = Gibco DNA and ASGR2-1. Lane 5 = Sisma DNA and ASGR2-1. Lane 6 = Mappmg panel human DN.A and ASGR2- 1. Lane7 = TEandCDMPl-l. Lane 8 = Gibco DNA a^nd CDMPl-1. Lane 9 = Sigma DNA andCDMPl-1. Lane 10 = Mapping panel human DNA and CDMPl- 1. Lane 11 = Gibco primer mix and DN.A. 40 Fig. 9 - Preliminaiy PCR of ASGR2-1 ( 146bp) with decreased stringency by decreasing the temperature and prehnunar\- PCR of RMS A (249bp). Lane 1 and 12 = lambda Hin D III DNA. Lane 2 = PhiX Hue III DNA. Lane 3 = TE and RMSA. Lane 4 = Sigma DNA and RMSA. Lane 5 = Mapping panel human DNA and RMSA. Lane 5 = Gibco DN.A and RMSA. Lane7 = TEand ASGR2-I. Lane 8 = Sigma DNA and ASGR2-1. Lane 9 = Mappmg panel human DN.A and ASGR2-1. Lane 10 = Gibco DNA and .A.SGR2-1. Lane I 1 = Gibco primer mi.\ and DNA. Fig. 10 - Unsuccessful chromosome assignment of CDMPl-2 l205bpi. No bands delected. Lanes 1 and 12 = lambda Hin D III DN.A. Lane 2 = PhiX Hue III DN.A. Lanes 3 - 6 = Pools 1 - 6 and CDMPl-2. Lane 7 = Mapping panel human DN.A and CD\IPl-2. Lane 8 - Mappinsj panel mouse DN.A and CDMPl-2. Lane 9 = Mapping panel Chinese hamster DN.A and CD.\IPl-2. Lane 10 = TE and CDMPl-2. Lane 1 1 = Gibco primer mix and DNA. Fig. 1 1 - Chromosomal pool assignment of CDMPl-2 (205bp) to pool 2. containing chromosomes 20 and 21. Lane I and 12 - lambda Hin D III DNA. Lane 2 - phiX Hue III DN.-X. Lane 3 = Pool 1. Lane 4 = Pool 2. Lane 5 = Pool 3. Lane 6 = Pool 4. Lane 7 = Pool 5. Lane 8 = Pool 6. Lane 9 = Pool 7. Lane 10 = Pool 8. Lane 1 1 = Pool 9. 43 Fig. 12 - Chromosome assignment of CDMP 1-2 (205bp) to chromosome 20. Lanes 1 and 1 r= lambda Hin D III DNA. Lane 2 = PhiX Hue III DNA. Lane 3 = chromosome 20 and CDMPl-2. Lane 4 = chromosome 21 and CDMP 1-2. Lane 5 = TE and CDMP 1-2. Lane 6 = Sigma DNA and CDMPl-2. Lane 7 = Mapping panel human DNA and CD.\IPl-2. Lane 8 = Mapping panel mouse DNA and CDNIPl-2. Lane 9 = Mappins panel Chmese hamster DNA and CDMPl-2. Lane 10 = Gibco DNA and CDMPl-2. 44 Fie. 13 - Chromosome assienment of CDMPl-2 (205bp) to chromosome 20. Lanes 1 and \0 = lambda Hin D III DNA. Lane 2 = IKh ladder. Lane 3 = TE and CD\lPl-2. Lane 4 = chromosome 2 and CDMPl-2. Lane 5 = chromosome 20 and CDMPl-2. Lane 6 = Mappms panel human DNA and CDMPl-2. Lane 7 = Mapping panel mou.se D.\A and CDMPr-2. Lane 8 = Mappmg panel Chinese hamster DNA and CD.MPl-2. Lane 9 = Gibco primer mix and DNA. ^aJ'>/ i^arh^i- , 4/0- A-HiaAIB: 2.^ l\c&l<,dob,r- "^--^If J-TE '+-ci\o>rO»-2- S'^cj.fo.^tifi i>-t^?y\::y^- ^^^?;^.^ - ' / 3-cA*^. 1- /