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Bioinformatics to Study Alu

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					Bioinformatics to Study Alu-PV92 Marker

1. Go to http://bioinformatics.dnalc.org/pv92/ then select Protocol, then select Bioinformatics, click NEXT (2
  times)

2. Highlight the primer set sequence and copy

3. Go to NCBI—Blast then select Nucleotide BLAST.

4. Paste the primer set into the ENTER QUERY SEQUNCE. Remove any non-nucleotide characters

5. Select parameters for search CHOOSE SEARCH SET ( o Others)
                                PROGRAM SELECTION ( o Somewhat similar sequences (blastn))
6. Select BLAST

7. You will receive the GRAPHIC SUMMARY……Note there is 1 match with 100% match

8. Go to the DESCRIPTION….note the first 3 hits

9. Complete the following for the first 4 matches

  Accession             Description                               E value               Max ID
 AC0090283                                                         0.002                100%


Note E value = Expectation value is the number of alignments with this primer sequences that would be
 expected to occur by chance. The lower E value = a higher probability the primers are related to this sequence
 of DNA
Note this sequence on Chromosome 16—Expect this to be an important match

10. Scroll to Alignment Section to see where the two primers have matched the DNA sequence

        (Primer )       Query 26 G----------etc….
        (DNA Seq )     Sbjcts 57137 G----------etc…


        (Primer )       Query 1 G----------etc….
        (DNA Seq )     Sbjcts 56722 G----------etc…

                         Thus the DNA sequence targeted by these primers is      57137
                                                                                - 56722
                                                                                    415
                                                    length of fragment +1




11. Click on Accession # AC0090283 to get data sheet

12. Go to sequence at bottom of data sheet and find the numbers 56722 to 57137 and HIGHLIGHT and COPY.

13. Open a new BLAST search and paste the 415 bp fragment sequence into the ENTER QUERY SEQ box.
14. Do a BLAST search with NUCLEOTIDE COLLECTION and blastn

Note the TOP 4 HITS under descriptions

  ACCESSION              Description    Max Score          Total             Q Co             E value

1. AC009028.3                                                                                 (0.0)

2. AF 302689.1

3. M57427.1

4. AL 390856.9

15. Why does the 1st hit have an E value of zero?________________________________________

16. Now focus on the hit named “Human Alu repeat.” This is the Alu insertion at PV92.

        a. Follow the Accession link, then click on repeat_region70..76/rpt_family="Alu" in the Features section.
           What do you notice about the 3’ end of the Alu repeat?



        b. Also in the Features section, look at the “insertion target sequence” on either side of the Alu repeat.
           What appears to be going on?

17. What is the length of the Alu inserted at PV92?



18. If you assume that the amplicon in Part I is the – allele, what is the length of the + allele?



19. Now look carefully at the hit named "Homo sapiens isolate BAS101 AluPV92 repeat sequence." Examine the
  Features and follow links. What is going on here? How are the three hits related to one another?



20. Return to the NCBI home page, then click on Map Viewer located in the Hot Spots column on the right.

21. Find Homo sapiens (humans) in the table to the right and click on the “B” icon under the Tools header. If
  more than one build is displayed, select the one with the highest number, as this will be the most recent
  version.

22. Paste the 416-bp amplicon (from Part I) into the search window. (Primers usually are not long enough to
  produce a result in the map BLAST.)

23. Select BLASTN from the drop-down menu under Program and click on Begin Search.
24. Click on View report to retrieve the results.

25. Click on [Human genome view] in the list of Other reports at the top of the page to see the chromosome
  location of the BLAST hit. On what chromosome have you landed?

7. Click on the marked chromosome number to move to the PV92 locus. Click on the small blue block labeled
  Genes seq to display genes. The 416-bp amplicon (red) occupies the whole field of the default view. What can
  you say about the gene that contains the amplicon? Click on the name under the Symbol track, and then
  follow links to find out.

8. Use the zoom out toggle on the left to get a better perspective on the CDH13 gene. Introns and noncoding
  sequences are denoted by a thin line, while exons are denoted by thick bar.

a. Determine the size of the CDH13 gene using the map coordinates to the left of the contig map.



b. How many introns and exons does CDH13 gene have?



c. Where in the CDH13 gene is PV92 Alu inserted: an exon or intron?



d. How does this explain the fact that the PV92 insertion is believed to be neutral – to have no phenotypic
  effect?

				
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posted:10/10/2011
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