Bioinformatics to Study Alu

fabulousgalaxyBiotechnology

Oct 1, 2013 (3 years and 8 months ago)

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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 si
milar 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 val
ue



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 seq
uence
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 numbe
rs 56722 to 57137 and HIGHLIGHT and COPY.

13.

Open a new BLAST search and paste the 415 bp fragment s
equence 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 1
st

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?

1
7. 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 t
he
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.

2
1
. 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 w
ill be the most recent
version.

22
. Paste the 416
-
bp amplicon (from Part I) into the search window. (Primers usua
lly 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 repo
rts 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 4
16
-
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 bett
er 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 ex
ons 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?