BLAST searches and related applications are essential for analyzing gene and protein sequences. Define BLAST, describe basic features of this bioinformatics tool, and give an example of information provided by a BLAST search.

What are community-based genetic screening programs? What is the intent of such screening programs? Why are members of specific communities or populations offered the chance to participate in such programs?

What is bioinformatics, and why is this discipline essential for studying genomes? Provide two examples of bioinformatics applications.

You are designing algorithms for the bioinformatic prediction of gene sequences. How might algorithms differ for predicting genes in bacterial versus eukaryotic genomic sequence?

Do you think it is important that participation in community-based genetic screening be entirely voluntary? Why or why not?

Go to http://blast.ncbi.nlm.nih.gov/Blast.cgi and follow the links to nucleotide BLAST. Type in the sequence below; it is broken up into codons to make it easier to copy.

5' ATG TTC GTC AAT CAG CAC CTT TGT GGT TCT CAC CTC GTT GAA GCTTTG TAC CTT GTT TGC GGT GAA CGT GGT TTC TTC TAC ACT CCT AAG ACT TAA 3'

As you will note on the BLAST page, there are several options for tailoring your query to obtain the most relevant information. Some are related to which sequences to search in the database. For example, the search can be limited taxonomically (e.g., restricted to mammals) or by the type of sequences in the database (e.g., cDNA or genomic). For our search, we will use the broadest database, the 'Nucleotide collection (nr/nt).' This is the nonredundant (nr) database of all nucleotide data (nt) in GenBank and can be selected in the 'Database' dialogue box. Other parameters can also be adjusted to make the search more or less sensitive to mismatches or gaps. For our purposes, we will use the default setting, which is automatically presented. Press 'BLAST' to search. What can you say about the DNA sequence?

In the course of the Drosophila melanogaster genome project, the following genomic DNA sequences were obtained. Try to assemble the sequences into a single contig.

5' TTCCAGAACCGGCGAATGAAGCTGAAGAAG 3'

5' GAGCGGCAGATCAAGATCTGGTTCCAGAAC 3'

5' TGATCTGCCGCTCCGTCAGGCATAGCGCGT 3'

5' GGAGAATCGAGATGGCGCACGCGCTATGCC 3'

5' GGAGAATCGAGATGGCGCACGCGCTATGCC 3'

5' CCATCTCGATTCTCCGTCTGCGGGTCAGAT 3'

Go to the URL provided in Problem 14, and using the sequence you have just assembled, perform a blastn search in the 'Nucleotide collection (nr/nt)' database. Does the search produce sequences similar to your assembled sequence, and if so, what are they? Can you tell if your sequence is transcribed, and if it represents protein-coding sequence? Perform a tblastx search, first choosing the 'Nucleotide collection (nr/nt)' database and then limiting the search to human sequences by typing Homo sapiens in the organism box. Are homologous sequences found in the human genome? Annotate the assembled sequence.

DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. Approximately what length is the DNA region protected by RNA pol II and TFIIs?

DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. What additional genetic experiments would you suggest to verify that this region of cloned DNA contains a functional promoter?