CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1

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1 CAP BIOINFORMATICS Su-Shing Chen CISE 10/5/2005 Su-Shing Chen, CISE 1

2 Genomic Mapping & Mapping Databases High resolution, genome-wide maps of DNA markers. Integrated maps, genome catalogs and comprehensive databases linking positional and functional data. A researcher can focus on a region by searching public mapping databases rather than performing lab experiments. 10/5/2005 Su-Shing Chen, CISE 2

3 Mapping and Sequencing Entire chromosome sequences are the whole space of genetic information. Known shorter sequences are markers and gene-based tags, such as expressed sequence tags (ESTs) and sequencetagged sites (STSs), gene sequences, prefinished DNA clone sequences, and completed genomic sequence tracts. 10/5/2005 Su-Shing Chen, CISE 3

4 Orientation of Chromosome Orders Maps are linear arrangements of gene markers or gene tags. Maps serve as a scaffold for orienting, directing, and troubleshooting sequencing projects. NCBI BLAST and electronic PCR (e- PCR) for finding marker/sequence identities. 10/5/2005 Su-Shing Chen, CISE 4

5 Genomic Map Elements DNA markers uniquely identifiable segments of DNA, nucleotide bases. Markers are detected by PCR or hybridization. Polymorphic or monomorphic. PCR based markers are sequencetagged sites (STS s). They are uniquely PCR amplified by its primer sequences. 10/5/2005 Su-Shing Chen, CISE 5

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8 Hybridization: creating a hybrid strand of DNA/RNA The two strands of a DNA molecule are denatured by heating to about 100 C = 212 F (a to b). At this temperature, the complementary base pairs that hold the double helix strands together are disrupted and the helix rapidly dissociates into two single strands. The DNA denaturation is reversible by keeping the two single stands of DNA for a prolonged period at 65 C = 149 F (b to a). This process is called DNA renaturation or hybridization. 10/5/2005 Su-Shing Chen, CISE 8

9 Hybridization: creating a hybrid strand of DNA/RNA Similar hybridization reactions can occur between any single stranded nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the renaturation process, the RNA competes with the coding DNA strand and forms double-stranded DNA/RNA hybrid molecule (c to d). These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe. 10/5/2005 Su-Shing Chen, CISE 9

10 PCR (Polymerase Chain Reaction) A DNA synthesis technique that amplifies specific regions of DNA defined by complementary sequences of two specific primers. The primers are short single stranded DNA molecules. In cycles, the DNA fragments increase exponentially. 10/5/2005 Su-Shing Chen, CISE 10

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12 PCR Polymerase chain reaction (PCR), is a common method of creating copies of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of molecules. In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests. 10/5/2005 Su-Shing Chen, CISE 12

13 Polymorphic Markers Markers have individual sequence variations. For construction of genetic linkage maps. A polymorphism has alleles in a population. Hybridization polymorphism restriction (enzyme) fragment length polymorphism (RFLP), VNTR (Variable Number of Tandem Repeats). PCR polymorphism microsatellites, short tandem repeats, single nucleotide polymorphisms (SNP). 10/5/2005 Su-Shing Chen, CISE 13

14 DNA Clones DNA Vectors Reproduce, propagate DNA fragments. 1987, YAC (Yeast Artificial Chromosome) libraries, 2 Mb fragments, unstable and chimerism. BAC (Bacterial Artificial Chromosome) libraries, Kb fragments, high DNA yields and integrity. DNA fingerprinting determines insert overlaps and constructs clone contigs. 10/5/2005 Su-Shing Chen, CISE 14

15 Various Maps Cytogenetic maps markers are localized that can be directly imaged. Genetic linkage maps polymorphic markers are ordered on recombination. Radiation hybrid maps - chromosome breakage induced by lethal doses of radiation to a donor cell line. Transcript maps transcribed sequences and diseased genes. Include EST s expressed sequences. Physical maps - genetic distances. STS s. Comparative maps conserved chromosome segments across different species. Integrated maps - 10/5/2005 Su-Shing Chen, CISE 15