Gtatgcagaaaatcttagagtgtcccatctggtaagtcagc

W S MYMSKKRWWS SMR ■ Figure 10-14 187 delAG mutation in the BRCA1 gene. This heterozygous dinucleotide deletion Is evident In the lower panel where, at the site of the mutation, two sequences are overlaid: the normal sequence and the normal sequence minus two bases.

Table 10.2 Software Programs Commonly Used to Analyze and Apply Sequence Data

Software

Name

Application

BLAST Basic Local Alignment Search Tool

GRAIL Gene Recognition and Assembly Internet

Link

FASTA FAST-All derived from FAST-P (protein)

and FAST-N (nucleotide) search algorithms Phred Phred

Compares an input sequence with all sequences in a selected database

Finds gene-coding regions in DNA sequences

Rapid alignment of pairs of sequences by sequence patterns rather than individual nucleotides

Reads bases from original trace data and recalls the bases, assigning quality values to each base

Continued on following page

Table 10.2 Software Programs Commonly Used to Analyze and Apply Sequence Data (continued)

Software

Name

Application

Polyphred Phrap

TIGR Assembler

Factura

SeqScape

Assign Matchmaker

Polyphred

Phragment Assembly Program

The Institute for Genomic Research

Factura

SeqScape

Assign Matchmaker

Identifies single nucleotide polymorphisms (SNPs) among the traces and assigns a rank indicating how well the trace at a site matches the expected pattern for an SNP Uses user-supplied and internally computed data quality information to improve accuracy of assembly in the presence of repeats Assembly tool developed by TIGR to build a consensus sequence from smaller-sequence fragments Identifies sequence features such as flanking vector sequences, restriction sites, and ambiguities. Mutation and SNP detection and analysis, pathogen subtyping, allele identification, and sequence confirmation Allele identification software for haplotyping Allele identification software for haplotyping the 3' end of the primer, DNA polymerase extends the primer. Pyrophosphate (PPi) is released with the formation of the phosphodiester bond between the dNTP and the primer. The PPi is converted to ATP by sulfurylase in the presence of APS. The ATP is used to generate a luminescent signal by luciferase-catalyzed conversion of luciferin to oxyluciferin. The process is repeated with each of the four nucleotides again added sequentially to the reaction. The generation of a signal indicates which nucleotide is the next correct base in the sequence. Results from a pyrosequencing reaction consist of single peaks of luminescence associated with the addition of the complementary nucleotide. If a sequence contains a repeated nucleotide, for instance, GTTAC, the results would be: dG peak, dT peak (double the height of the dG peak), dA peak, dC peak.

Pyrosequencing is most useful for short- to moderate-sequence analysis. It is therefore used mostly for mutation or single nucleotide polymorphism (SNP) detection and typing rather than for generating new sequences. It has been used for applications in infectious disease typing 910 and HLA typing.11

Bisulfite DNA Sequencing

Bisulfite DNA sequencing, or methylation-specific sequencing, is a modification of chain termination se-

Advanced Concepts

Pyrosequencing requires a single-stranded sequencing template. Methods using streptavidin-conjugated beads have been devised to easily prepare the template. First the region of DNA to be sequenced is PCR-amplified with one of the PCR primers cova-lently attached to biotin. The amplicons are then immobilized onto the beads and the nonbiotinylated strand denatured with NaOH. After several washings to remove all other reaction components, the sequencing primer is added and annealed to the pure single-stranded DNA template.

quencing designed to detect methylated nucleotides.12,13 Methylation of cytosine residues in DNA is an important part of regulation of gene expression and chromatin structure (see Chapter 2). Methylated DNA is also involved in cell differentiation and is implicated in a number of diseases, including several types of cancer.

For bisulfite sequencing, 2-4 ^g of genomic DNA is cut with restriction enzymes to facilitate denaturation. The enzymes should not cut within the region to be sequenced. The restriction digestion products are resolved on an agarose gel, and the fragments of the size of inter-

Step 1

Polymerase

Step 2 Luciferin

Oxiluciferin

Step 2 Luciferin

Oxiluciferin

Step 3

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