SOLICITATION NOTICE
B -- Clinical Research Support Services
- Notice Date
- 9/14/2006
- Notice Type
- Solicitation Notice
- NAICS
- 541990
— All Other Professional, Scientific, and Technical Services
- Contracting Office
- Department of Health and Human Services, National Institutes of Health, National Heart, Lung and Blood Institute, Rockledge Dr. Bethesda, MD, Office of Acquisitions 6701 Rockledge Dr RKL2/6100 MSC 7902, Bethesda, MD, 20892-7902
- ZIP Code
- 20892-7902
- Solicitation Number
- NHLBI-PB-(HL)-2006-302-DDC
- Response Due
- 9/25/2006
- Archive Date
- 10/10/2006
- Description
- THIS IS A NOTICE OF INTENT, NOT A REQUEST FOR A PROPOSAL. A SOLICITATION DOCUMENT WILL NOT BE ISSUED AND PROPOSALS WILL NOT BE REQUESTED. The National Heart, Lung, and Blood Institute?s (NHLBI), Office of Acquisitions (OA), intend to negotiate and award a purchase order on a noncompetitive sole source basis to Children?s Hospital & Research Center Oakland, 5700 Martin Luther King Jr. Way, Oakland, CA., 94609, to provide BAC Library Construction and Screening of Genomic DNA from ENU mouse mutants. The sole source determination is based upon the requirement for making BAC Libraries and retrieval of BAC Clones Spanning Critical Region containing ENU induced Mouse mutations. NHLBI is interested exploring improved mapping & sequencing procedures for identifying mutations in ethylnitrosourea (ENU) mutagenized mice with congenital heart defect. Dr. Pieter de Jong, is the head of the BAC/PAC Resource Center (BPRC) of Children?s Hospital Research Center Oakland was selected based on the fact he is well recognized as the world?s leading expert in making and screening of BAC libraries. He has generated many of the BAC Libraries used in the Human Genome Project, and nearly all of the existing BAC libraries from inbred mouse strains in the Mouse Genome Project. In addition he has also developed a very high throughput pipeline for screening BAC libraries involving the unique assembly of macroarrays, and the continue use of the BAC Libraries in the Research Protocol. Based upon the knowledge, skills, and expertise possessed by Dr. Pieter de Jong, an award to any other source would affect the Institute?s anticipated outcome based upon Dr. Jong?s experience, and would not provide any cost savings to the Government. Statement of Work: Making BAC Libraries and Retrieval of BAC Clones Spanning Critical Region Containing ENU Induced Mouse Mutations. Summary: NHLBI is interested exploring improved mapping & sequencing procedures for identifying mutations in ethylnitrosourea (ENU) mutagenized mice with congenital heart defect and is seeking to subcontract part of the work. Many recessive mutations in different mutant mice pedigrees have been mapped through genetic crosses to distinct genomic intervals spanning between one and five mega basepairs. The embryos homozygous for the mutation will be the source of DNA for the sequencing project. One or more frozen embryos per mutant pedigree will be available to the contractor. The contactor will isolate high molecular weight DNA from these embryos to create one DNA clone collection per mutant pedigree and will screen the DNA clone collection for all clones from the genomic interval containing the defective gene. This region will from here-on be referred to as the critical region. Following the identification and isolation DNA clones derived from the critical region, these clones will be characterized further by sequencing of the paired ends of the cloned DNA fragments. The paired-end sequences will be matched to the mouse genome as available through public databases. The goal is to identify a minimal set of DNA clones spanning all or most (>98%) of the critical region. These isolated minimal set of clones will be delivered to NHBLI and will serve as the substrates for high-throughput genomic sequencing of the critical region. DEFINITIONS: DNA Clones: E.coli clones containing large DNA fragments (average 150 kilo base pairs) of mouse genomic DNA. These fragments will have been cloned into a low-copy number ?Bacterial Artificial Chromosome? (BAC) vector. BAC vector: Many versions of BAC vectors now exist. The contractor should have extensive track-record with cutting-edge BAC vectors and their use to create mouse BAC libraries. BAC library: A collection of DNA clones created from a single DNA source, each derived from a single, unique cloning event in E.coli. The cloning process should approximate as much as possible a random process. In this case, each library will have been generated from pooled homozygously-mutated embryos from a single mutant pedigree. Arrayed BAC library: Clones, derived from unique cloning events, will be grown in spatial separation. To this aim, the freshly-transformed E.coli cells will have to be separated prior to first cell division. This is needed to ensure that all BAC clones are independent. The process requires colony-picking robots to move E.coli colonies from Petri dishes into separate wells of 384-well dishes. The wells contain growing media with a proper antibiotic (in most cases, chloramphenicol) and supplemented with glycerol (for cryo-preservation of the cultures). BAC library size: The size of a BAC library is defined by the level of redundancy within the random clone collection. A ?tenfold redundant? BAC library will be needed for this process to improve the odds that all sequences from the critical region are present at least once in the clone collection. On average, each sequence element will be present 10 times in independent BAC clones. This high level of redundancy is required for two reasons: 1) to minimize or avoid any gaps in the ?critical region? due to sequences missing from the BAC library, and 2) to maximize the number of options for selecting BAC clones which minimally overlap with other BAC clones with respect to sequence content. This is important because the cost of sequencing is proportional to the number of BAC clones submitted to the sequencing pipeline. Extensive overlaps between BAC clones add significantly to the downstream cost of sequencing the entire critical region. BAC Array size: The total number of microtiter dishes corresponding to a single copy of the BAC library. For a tenfold redundant mouse BAC library, about 500-600 microtiter dishes will be needed. BAC library quality: The quality of a BAC library is defined by a set of different parameters: 1) Randomness of the cloning process: partial-digestion with a restriction enzyme (EcoRI) will be required to generate the random fragments in advance of cloning, 2) Average insert sizes of the cloned DNA fragments (average 150 kilo base pairs (kbp) or larger will be required , 3) Minimal levels of cloning artifacts, in particular those which result in chimeric clones containing multiple unrelated fragments in a single BAC. The contractor will need to have a proven track record of creating BAC libraries with chimera levels below 1%. 4) Minimal levels of well without any clones. Less than 10% empty well required. BAC library screening: The BAC library will need to be screened to identify BAC clones containing sequences derived from the critical region. This will need to be done by DNA hybridization of complex mixtures of probes, to membranes containing macro-arrays representative of the arrayed BAC library. The contractor will need to have a proven track record creating high quality macro-arrays for hybridization screening and a record of using these for screening BAC libraries with complex probe mixtures. Macro-arrays: Membranes containing large numbers of DNA spots derived from BAC clones. The spots are placed in regular, organized patterns and each spot can be traced back to a specific BAC clone archived in a freezer as part of an arrayed BAC library. Each membrane should represent all clones contained in 48 microtiter dishes of the BAC library. The entire library might be represented by as much as 10 such macro-arrays. Probe mixtures: In order to identify all clones through a single hybridization screening, radioactively-labeled probes representing the critical region will be pooled together. Probes will need be selected at regular (50 kbp) intervals along the critical region and will all have a similar composition (but distinct sequence). Intervals of approximately 50 kbp will ensure that each BAC from the critical region will likely be positive for several probes to minimize false-negative rates. Probes: To permit probes to be mixed in large numbers, they will need to behave at similar hybridization stringency to avoid ?weaker probes? being masked by better hybridizing probes. The probe will need to be designed with this requirement in mind. Overgo probes are synthetic DNA probes derived from 3?overlapping 24-mers with an 8 nucleotide overlap. These probes are labeled by extending the 3?ends using a DNA polymerase and 32P-dNTPs. Overgo probes are designed to have the same melting point and can thus be co-hybridized at the same stringency. Twenty probes are required per Mbp of genomic DNA. Up to 150 probes (8 Mbp) can be pooled for a successful screen. BAC re-arraying: BAC clones are identified in the screening process through positive hybridization spots on the macro arrays. These BACs are considered candidate clones containing sequences derived from the critical region. Twenty probes are required per Mbp of genomic DNA. Up to 150 probes (8 Mbp) can be pooled for a successful screen. BAC re-arraying: BAC clones are identified in the screening process through positive hybridization spots on the macro arrays. These BACs are considered candidate clones containing sequences derived from the critical region. BAC characterization: BAC clones from the critical region will need to be confirmed and defined. The BAC clones will need to be submitted to a sequencing center or company for determining the sequences at the ends of inserts. The paired end sequence provide crucial information defining the size and chromosomal position of the clones. The contractor will select a minimal number of BAC clones spanning the region. It is likely that some sequences are not present either due to incompatibility for cloning in E.coli or for statistical reasons. The missing sequences should ideally not encompass more than 4% of the critical region. To eliminate false-negatives from the screening process, the macro arrays might be screened again with a limited set of probes neighboring the clone gaps. The BAC clones from the minimal set will be characterized for size using pulsed-field electrophoresis. The size of each BAC should match the predicted size. The predicted size is determined from the distances spanned by the paired BAC-end sequences relative to the mouse genome assembly in the public databases. PROCESS DESCRIPTION: The contractor will receive a number of snap-frozen embryos for each of the mutant pedigrees to be processed. The embryos will be grounded to powder using a pestle and mortar under liquid nitrogen. The powder will be suspended in a chromatin-extraction buffer to minimize shearing and allow the DNA to be maintained in high-molecular weight form. The suspension will be mixed with liquefied agarose and then cooled down to solidify the agarose in a gel mold to create plugs of a defined size. The plugs with the agarose-embedded chromatin will be treated with detergents and proteases to release the matrix-stabilized DNA. The DNA will susbsequently be treated with EcoRI restriction enzyme under partial-digest conditions, meaning that most EcoRI restriction sites will not be cut. The resulting DNA fragment mixture is size-fractionated using pulsed-field gel electrophoresis in a CHEF apparatus (BioRad). The appropriate size fractions are sliced from the gel, characterized for size and then used for ligation reactions with the BAC vector (pBAC-GK1.1). The transformation mixtures will be used to transform highly-electrocompetent (InVitrogen) DH10B E.coli cells (T1-phage resistant). Several hundred transformations will be required to generate a 10-fold redundant BAC library with average inserts of around 150 kbp. The freshly-transformed cells mixtures will be cryo-preserved pending characterization to determine suitability and compatibility with the required quality standards. The cells are than spread on big square Petri dishes (20 x 20 cm) to create colonies. The colonies are transferred into separate wells of 384-well dishes, using a Q-pick colony arraying robot. Following overnight growth of the well cultures, the dishes are stored in the freezer until further use. To create the macro arrays, the 384-well dishes are thawed and processed in a filter-gridding robot to create a macro-arrays of >18,000 colonies per membrane. Following overnight growth of the colonies, the membranes are processed to release the DNA and crosslink to the membranes to create the DNA spot array. The spot arrays (about 10 different macro-arrays per BAC library) will be screened by DNA hybridization using complex mixtures of radioactive overgo probes. DELIVERABLES: 1. One (or more) sets of BAC clones containing sequences from the critical regions from corresponding ENU mutant pedigrees. These BACs will on average have sizes of 150 kbp or better. The BAC clones will be contained in 384-well dish format and will be shipped on dry ice to NHLBI for further analysis. 2. Characterization data for these BAC clones and identification of a subset of BACs containing the minimal BAC tiling set. Period of Performance: The entire process will take a minimal of 3 months per mutant pedigree and may take up to one month longer and is contingent on several external factors. These factors include the quality of the DNA source material and the advance scheduling of the project (or lack there-off). Multiple projects may overlap with one month intervals. This acquisition is being conducted under simplified acquisition procedures and is exempt from the requirement of Federal Acquisition Regulations (FAR) Part 6, Competition Requirements. The North American Industry Classification System (NAICS) applicable to this requirement is 541990 with a size standard of $6.5 M. This notice of intent is not a request for competitive proposals. Interested parties may identify their interest and capabilities in response to this synopsis. The determination by the Government not to compete the proposed contract based upon responses to this notice is solely with the discretion of the Government. Comments to this announcement, referencing synopsis number NHLBI-PB(HL)-2006-302-DDC, may be submitted to the National Heart, Lung, and Blood Institute, Office of Acquisitions, Procurement Branch, 6701 Rockledge Drive, Suite 6042, Bethesda, MD 20892-7902, Attention: Deborah Coulter.
- Place of Performance
- Address: Bethesda, MD
- Zip Code: 20892-7902
- Country: UNITED STATES
- Zip Code: 20892-7902
- Record
- SN01143348-W 20060916/060914220357 (fbodaily.com)
- Source
-
FedBizOpps Link to This Notice
(may not be valid after Archive Date)
| FSG Index | This Issue's Index | Today's FBO Daily Index Page |