Chapter_9_-_Characterization_of_a_Cloned

Chapter_9_-_Characterization_of_a_Cloned - Characterization...

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Characterization of a Cloned Gene
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9.1 DNA sequencing 9.1.1 Principles of DNA Sequencing The most common method of sequencing is dideoxy terminator method or the Sanger method DNA synthesis requires annealing of a primer to the template strand Bases are added to the growing strand by covalent phosphodiester bond between 5’ P (on nucleotide to be added) and 3’-OH (group on the existing molecule) ddNTP has a 2’,3’-dideoxyribose sugar, lacking a 3’OH group in the 3’ position compared to dNTP (2’deoxyribose) Addition of ddNTP to the strand terminates DNA synthesis Figure 9.2 2’deoxyribose and 2’,3’ dideoxyribose Figure 9.3 Chain termination by ddNTP
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9.1 DNA sequencing 9.1.1 Principles of DNA Sequencing one base as a dideoxy-derivative such as ddGTP is included along with dGTP,dATP,dCTP,dTTP in the DNA sequencing reaction With some of the dGTPs replaced by ddGTP, DNA synthesis will proceed along a range of chain lengths and terminate with a ddGTP residue In manual sequencing, each of four lanes is spiked with a different ddNTPs one of the dNTPs is radioactively labeled, revealing chain length sizes as a pattern of black bands on X-ray film Figure 9.1 Determination of DNA sequence
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9.1 DNA sequencing  9.1.2 Automated Sequencing In automated sequencing, sequence is obtained by separation of the terminated extension products on a matrix-filled capillary the sequencing reaction mix contains the four ddNTPs conjugated with a unique fluorescent dye. Each dye when excited by a laser emits a specific fluorescence. This fluorescence is detected by a CCD and processed into an electropherogram or chromatogram ABI 3700 sequencer
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9.1.2 Automated sequencing  Sample chromatogram
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9.1.3 Extending the Sequence Typical sequencing reads are 600 to 800 bp If sequencing from a vector, the insert can be sequenced from both directions across the insert from universal forward/backward primers To read genes with long inserts, you can walk along the gene by sequencing from primers designed from the new sequence Figure 9.4 Chain termination by ddNTP
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9.1.4 Shotgun Sequencing; Contig Assembly Long fragments to be sequenced can be split into smaller fragments for sequencing, known as shotgun cloning and sequencing Random cloned fragments are sequenced, until a full length contig (contiguous sequence) has been assembled by sequencing overlapping fragments Figure 9.5 Shotgun cloning and sequencing Figure 9.6 Contig formation and joining
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9.2 Genbank Databank entries and annotation Entrez Gene ID number and official name refer to any human homolog of a gene (eg. Gene ID: 50688, Official Full name: calcium channel, voltage-dependent, beta 1 subunit) Accession number is an official number for any entry into the database (mRNA, genomic DNA, protein sequence); there may be dozens of accession numbers for one gene Annotations: organism, publication and source references, CDS (CoDing
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_9_-_Characterization_of_a_Cloned - Characterization...

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