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===[[QuBi/modules/biol302#Key Concepts2|Key Concepts]]===
===[[QuBi/modules/biol302#Key Concepts2|Key Concepts]]===
*Homology searching using BLAST
*Homology searching using BLAST ('''not Google''')
*Mammalian gene regulation
*Mammalian gene regulation


So far, you have excised this XbaI fragment:
So far, you have excised this XbaI fragment:
<div style="font-family:Monospace;line-height:1;width:550px;border-style:solid;border-width:1px;border-color:#AAAAFF;background-color:#EEEEFF;padding-left:5px;padding-right:5px;padding-top:0px;padding-bottom:0px;">
<div style="font-family:Monospace;line-height:1;width:550px;border-style:solid;border-width:1px;border-color:#AAAAFF;background-color:#EEEEFF;padding-left:5px;padding-right:5px;padding-top:0px;padding-bottom:0px;">
TCTAGATGCATTTACGAAGGAGACAGAAAACGTCTTTCGGCAATAGCTCTCAAATGCAAAACGACGTCGG
CTAGATGCATTTACGAAGGAGACAGAAAACGTCTTTCGGCAATAGCTCTCAAATGCAAAACGACGTCGG
CGAGCTGTCCCTTACCTGGAGGCCCGCAGGAGAAGCGCGGTGATCCGAGAGGGTCCCCCAGGGGTGTCCG
CGAGCTGTCCCTTACCTGGAGGCCCGCAGGAGAAGCGCGGTGATCCGAGAGGGTCCCCCAGGGGTGTCCG
GTCGGTCTCCCGCTCGCCCAGCAGACGGCTGCGGAAACGGGGCAGCGTTTAAATAACCCCAGCTGGAGAC
GTCGGTCTCCCGCTCGCCCAGCAGACGGCTGCGGAAACGGGGCAGCGTTTAAATAACCCCAGCTGGAGAC
ATGTCAGGACTTAGCTCCTCCGACAGCCGACGCCGGACGTGTCCCAACTTGACCAGCCCCACAGGAAGAG
ATGTCAGGACTTAGCTCCTCCGACAGCCGACGCCGGACGTGTCCCAACTTGACCAGCCCCACAGGAAGAG
CTGAGTCAACTCGGCCCAGCCCAGTCCCACCCGTCCCGGAAGCCGCATCCCGGCGAGTCCGGGACCAGGC
CTGAGTCAACTCGGCCCAGCCCAGTCCCACCCGTCCCGGAAGCCGCATCCCGGCGAGTCCGGGACCAGGC
ACCTGTCACCTCCTGGACCCCAGCAACGAGCCCAGCGCGACCCCGGAGCGGGCCCGAATTCTCTAGA
ACCTGTCACCTCCTGGACCCCAGCAACGAGCCCAGCGCGACCCCGGAGCGGGCCCGAATTCT
</div>
</div>
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# Copy and paste the above sequence into the "Enter Query Sequence" box
# Copy and paste the above sequence into the "Enter Query Sequence" box
# Scroll down to the bottom of the page and click "BLAST"
# Scroll down to the bottom of the page and click "BLAST"
# Wait for 10-30 seconds for the results to return ('''Be Patient'''). Once the result page is loaded, locate and copy the following information:
# Wait for 10-30 seconds for the results to return ('''Be Patient'''). Once the result page is loaded, locate and copy/write down the following information:
## Species and strain
## Species and strain
## Chromosome
## Chromosome
## Length of your query sequence
## Length of your query sequence
## Sequence identity, number of matched bases, and number of gaps between the matched sequences
## Sequence identity, number of matched bases, and number of gaps between the matched sequences
## Full name of the gene that matches your query
# Click the link for "5' side" (next to '''Features''') will bring you a standard GenBank file of this gene. Locate and copy the following structural information about this gene:
# Click the link for "5' side" gene will bring you a standard GenBank file of this gene. Locate and copy the following structural information about this gene:
## Gene accession (ID number)
## Gene accession (ID number)
## Total length of the gene
## Total length of the gene
## Number of introns
## Number of introns
## Which is the actual coding strand (mRNA analog): the above sequence itself or its reverse complement?
## Which is the non-template (mRNA analog) strand: the above sequence itself or its reverse complement? [Hint: note the word '''complement''' in mRNA and cDNA lines)
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===Identify functional element with literature search===
===Identify regulatory elements through literature search===


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<div style="font-family:Monospace;line-height:1;width:800px;border-style:solid;border-width:1px;border-color:#AAAAFF;background-color:#EEEEFF;padding-left:5px;padding-right:5px;padding-top:0px;padding-bottom:0px;">
[[File:Zauberman_fig2.png|800px]]
[[File:Zauberman_fig2.png|800px]]


[[PROPERLY CITE THIS]] ''Taken from '' A functional p53-responsive intronic promoter is contained within the human mdm2 gene. ''[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC307078/ Pubmed] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC307078/pdf/nar00014-0022.pdf PDF]''
''Source:'' Zauberman et al. "A functional p53-responsive intronic promoter is contained within the human mdm2 gene". Nucleic Acids Res. 1995 July 25; 23(14): 2584–2592. ''[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC307078/ Pubmed] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC307078/pdf/nar00014-0022.pdf PDF]''
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# Once the TATA box is located, use this sequence as the anchor point to locate other elements, including the two p53 Response Element (p53 RE) sites and the Exon 2
# Once the TATA box is located, use this sequence as the anchor point to locate other elements, including the two p53 Response Element (p53 RE) sites and the Exon 2
# Locate the EcoRI restriction site using the [http://tools.neb.com/NEBcutter2/ NEBcutter website]
# Locate the EcoRI restriction site using the [http://tools.neb.com/NEBcutter2/ NEBcutter website]
# What are the expected lengths of EcoRI-digested fragments if your clone is oriented correctly (i.e., upstream of the Luciferase gene)
# What are the expected EcoRI fragment sizes for the two possible orientations of your cloned DNA?
# What if your cloned fragment is in the opposite direction (i.e., the TATA box situates downstream of the luciferase gene)
# Which orientation would you expect to give higher luciferase expression?


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==[[QuBi/modules/biol302#Part 2: Identification of mdm2 Splice Variants Using BLAST|Part 2 (Extra Credit Assignment): Identification of mdm2 Splice Variants Using BLAST]]==
==[[QuBi/modules/biol302#Part 2: Identification of mdm2 Splice Variants Using BLAST|Part 2 (Extra Credit Assignment): Identification of mdm2 Splice Variants Using BLAST]]==
<div class="center">
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[[File:4seq.png|800px]]
[[File:4seq.png|800px]]


''A diagram of the MDM2 gene used in this exercise, along with its splice variants. By the end of this module you will create a similar diagram.''
A diagram of the MDM2 gene used in this exercise, along with its splice variants. By the end of this module you will create a similar diagram. ''Reference:'' Arva NC, Talbott KE, Okoro DR, Brekman A, Qiu WG, Bargonetti J. 2008. Disruption of the p53-Mdm2 complex by Nutlin-3 reveals different cancer cell phenotypes. Ethnicity and Disease. 18(S2):1-8.
</div>
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===[[QuBi/modules/biol302#Objectives|Key Concepts]]===
 
* Use BLAST ('''not Google''') to find matches of DNA and protein sequences
 
* Alternative splicing and isoforms of a single gene
===[[QuBi/modules/biol302#Objectives|Objectives]]===
You will use the following table for your exercise:
* Learn to use Genbank database and BLAST tool to analyze nucleotide sequences
* Use BLAST to identify
 
===[[QuBi/modules/biol302#Key Concepts|Key Concepts]]===
; Blast
; Genbank
; Annotation
; Accession Number
; Alternative Splicing
 
===[[QuBi/modules/biol302#Exercise|Exercise]]===
{| class="wikitable"
{| class="wikitable"
! Genbank Accession # !! cDNA Clone !! Description !! Cell Line !! Length (bp)
! Genbank Accession # !! cDNA Clone !! Description !! Cell Line !! Length (bp)
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| AF527840 || || Genomic DNA || || 34,088
| AF527840 || || Genomic DNA || || 34,088
|-
|-
| EU076746 || P2-MDM2-C1 || cDNA missing exons 5-9 & 11 || MANCA || 427
| EU076746 || P2-MDM2-C1 || cDNA  || MANCA || 427
|-
|-
| EU076747 || P2-MDM2-10 || cDNA missing exon 10 || ML-1 || 842
| EU076747 || P2-MDM2-10 || cDNA || ML-1 || 842
|-
|-
| EU076748 || P2-MDM2-C  || cDNA missing exons 5-9 || A876 || 505
| EU076748 || P2-MDM2-C  || cDNA  || A876 || 505
|-
|-
| EU076749 || P2-MDM2-FL || Full-length cDNA || SJSA-1 || 845
| EU076749 || P2-MDM2-FL || cDNA || SJSA-1 || 845
|-
|-
|}
|}
*[[NOTE: Maybe we should remove the "Description" section of this table and leave it up to the students to figure out which exons are missing]]


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; Explore the gene annotation for AF527840.
===Explore the GenBank file===
Sequences on [[genbank]] have both basic reference information (such as what the sequence is, what organism it came from, and bibliographical information) and sequence [[annotations]]. Some sequences are more richly annotated than others - it is up to researchers to annotate the sequences they generate, which requires extra work. For this exercise you will be working will a well-annotated sequence: [[accession number]] AF527840. Explore its annotation and use it to complete the following set of tasks.
#Search [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide GenBank] using the accession AF527840. Read the GenBank file and find out from the feature table how many introns and exons this sequence has according to the "mRNA" and "CDS" features.  
 
# DRAW a diagram of this gene using the information and coordinates listed in the annotation.  
# DRAW a diagram of this gene using the information and coordinates listed in the annotation. (Note: this is the bulk of the assignment and this diagram is needed for the last set of questions. Don't get lazy on this.)
##Label the top of the diagram with basic information, such as the gene's name and species information.
##Label the top of the diagram with basic information, such as the gene's name, organism, etc.. Someone should be able to pick up your diagram and know exactly what they're looking at.
##Label coordinates for introns, exons, 3'/5' UTRs,start-codon, and stop-codon coordinates.  
##Including introns, exons, 3'/5' UTRs, +1, and exact coordinates. (The mRNA annotation states which segments are used to create mRNA, and the CDS annotation states which parts code amino acids (CDS = coding sequence)).
##Draw the diagram mostly to scale. It does NOT have to be perfect, but make a reasonable effort. Put a scale bar and length markers on your drawing.
##Draw the diagram mostly to scale. It does NOT have to be perfect, but make a reasonable effort. Put a scale bar and length markers on your drawing.
# How does the sequence vary at positions X, X, and X for this gene? Do these change the AA for the resulting peptide?
# What kinds of repeat regions can be found in this gene?
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; Explore the graphical presentation of the gene to answer the following question.
===Explore the graphical presentation of the gene===
Genbank provides graphical representations of the sequences on its database: click the "Graphics" link below the sequence title, OR click "Display Settings" above the title, and choose "Graphics". Take a few minutes to explore this graphical browser and answer the following question:
Genbank provides graphical representations of the sequences on its database: click the "Graphics" link below the sequence title, OR click "Display Settings" above the title, and choose "Graphics". Take a few minutes to explore this graphical and compare it with your diagram.
 
----
# A question that can only be answered from looking at this graph.
===Use BLAST to determine which exons are used in the mRNA transcript===
# Another question that can only be answered from looking at this graph.
This is the most "bioinformatic" part of the assignment. Blast one of the mRNA sequences (EU076746, EU076747, EU076748, EU076749) against the main sequence (AF527840) and use the results to answer the following questions. Suggested procedures:
 
# Go to the [http://www.ncbi.nlm.nih.gov/BLAST/ NCBI BLAST] website
# Click the link “Align two (or more) sequences using BLAST (bl2seq)” under “Specialized BLAST” (near the page bottom)
# In the “Sequence 1” text box, type “AF527840” (the accession for the genomics).  Fill in “from 1” and “to 34088”. In the “Sequence 2” text box, type in "EU076748" (or other cDNA accession in the table). Fill in the “from” box with 1 and the “to” box with 505.
# Click “Align”.  You should get a “Blast Result” output page.
# Interpret your results:
## Which exons are present and which ones are absent in EU076746, EU076747, EU076748, EU076749? (Hint: Refer to the mRNA join statement).
## Explain the following BLAST terms: “Expect” (e-value), “Identities”, “Gap”, “Strand”
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; Use BLAST to determine which exons are used in the mRNA transcripts.
This is the most "bioinformatic" part of the assignment. Blast ALL FOUR of the mRNA sequences (EU076746, EU076747, EU076748, EU076749) against the main sequence (AF527840) and use the results to answer the following questions. If you labeled your diagram well (with coordinates!), this task should go by quickly.
# Which exons are used to create EU076746, EU076747, EU076748, EU076749?
# Do the BLAST search results corresponding to exons ''exactly'' match the start/end positions of the exons as labeled in your diagram? If not, what is the most likely reason for this?
# Do any of the BLAST results match regions outside of exons? If so, what regions?
<!-- ===[[QuBi/modules/biol302#Exit_Questions|Exit Questions]]=== -->

Latest revision as of 14:50, 23 March 2013

BIOL 302 Lab (Bioinformatics Exercises)

Research in molecular genetics requires effective use of bioinformatic tools to analyze and understand the genetic materials being worked with. The following exercises will expose you to real-world scenarios and introduce you to the methods and tools you can use to solve these problems.

Part 1: Cloning of murine mdm2 gene sequence to study cis acting DNA elements

Key Concepts

  • Homology searching using BLAST (not Google)
  • Mammalian gene regulation

So far, you have excised this XbaI fragment:

CTAGATGCATTTACGAAGGAGACAGAAAACGTCTTTCGGCAATAGCTCTCAAATGCAAAACGACGTCGG CGAGCTGTCCCTTACCTGGAGGCCCGCAGGAGAAGCGCGGTGATCCGAGAGGGTCCCCCAGGGGTGTCCG GTCGGTCTCCCGCTCGCCCAGCAGACGGCTGCGGAAACGGGGCAGCGTTTAAATAACCCCAGCTGGAGAC ATGTCAGGACTTAGCTCCTCCGACAGCCGACGCCGGACGTGTCCCAACTTGACCAGCCCCACAGGAAGAG CTGAGTCAACTCGGCCCAGCCCAGTCCCACCCGTCCCGGAAGCCGCATCCCGGCGAGTCCGGGACCAGGC ACCTGTCACCTCCTGGACCCCAGCAACGAGCCCAGCGCGACCCCGGAGCGGGCCCGAATTCT

Gene identification using genome BLAST

  1. Go to the NCBI-BLAST website at NCBI/BLAST Home Page
  2. What is BLAST? Find the expanded answer by clicking on "more"
  3. Since BLAST finds matches (homologous sequences) between biological sciences, it needs a "query" sequence as input as well as a "database" to search against. To find the matches to the above sequence, what would be your "query" sequence and what would be your "database"?
  4. Start BLASTing against the mouse genome by clicking "Mouse" under "BLAST Assembled RefSeq Genomes"
  5. Copy and paste the above sequence into the "Enter Query Sequence" box
  6. Scroll down to the bottom of the page and click "BLAST"
  7. Wait for 10-30 seconds for the results to return (Be Patient). Once the result page is loaded, locate and copy/write down the following information:
    1. Species and strain
    2. Chromosome
    3. Length of your query sequence
    4. Sequence identity, number of matched bases, and number of gaps between the matched sequences
  8. Click the link for "5' side" (next to Features) will bring you a standard GenBank file of this gene. Locate and copy the following structural information about this gene:
    1. Gene accession (ID number)
    2. Total length of the gene
    3. Number of introns
    4. Which is the non-template (mRNA analog) strand: the above sequence itself or its reverse complement? [Hint: note the word complement in mRNA and cDNA lines)

Explore pGL2 vector

This sequence has been cloned into the NheI site in the pGL2basic vector:

PGL2vector-map.png Source: Promega

  1. Identify the location where you have cloned your fragment
  2. What are the two possible directions (with respect to the luciferase gene) you may have cloned your fragment?
  3. From the PDF file, find the location of the EcoRI site

Identify regulatory elements through literature search

Zauberman fig2.png

Source: Zauberman et al. "A functional p53-responsive intronic promoter is contained within the human mdm2 gene". Nucleic Acids Res. 1995 July 25; 23(14): 2584–2592. Pubmed PDF

  1. Identify the TATA box in the above sequence by looking for AT-rich regions
  2. Once the TATA box is located, use this sequence as the anchor point to locate other elements, including the two p53 Response Element (p53 RE) sites and the Exon 2
  3. Locate the EcoRI restriction site using the NEBcutter website
  4. What are the expected EcoRI fragment sizes for the two possible orientations of your cloned DNA?
  5. Which orientation would you expect to give higher luciferase expression?

Part 2 (Extra Credit Assignment): Identification of mdm2 Splice Variants Using BLAST

4seq.png

A diagram of the MDM2 gene used in this exercise, along with its splice variants. By the end of this module you will create a similar diagram. Reference: Arva NC, Talbott KE, Okoro DR, Brekman A, Qiu WG, Bargonetti J. 2008. Disruption of the p53-Mdm2 complex by Nutlin-3 reveals different cancer cell phenotypes. Ethnicity and Disease. 18(S2):1-8.

Key Concepts

  • Use BLAST (not Google) to find matches of DNA and protein sequences
  • Alternative splicing and isoforms of a single gene

You will use the following table for your exercise:

Genbank Accession # cDNA Clone Description Cell Line Length (bp)
AF527840 Genomic DNA 34,088
EU076746 P2-MDM2-C1 cDNA MANCA 427
EU076747 P2-MDM2-10 cDNA ML-1 842
EU076748 P2-MDM2-C cDNA A876 505
EU076749 P2-MDM2-FL cDNA SJSA-1 845

Explore the GenBank file

  1. Search GenBank using the accession AF527840. Read the GenBank file and find out from the feature table how many introns and exons this sequence has according to the "mRNA" and "CDS" features.
  2. DRAW a diagram of this gene using the information and coordinates listed in the annotation.
    1. Label the top of the diagram with basic information, such as the gene's name and species information.
    2. Label coordinates for introns, exons, 3'/5' UTRs,start-codon, and stop-codon coordinates.
    3. Draw the diagram mostly to scale. It does NOT have to be perfect, but make a reasonable effort. Put a scale bar and length markers on your drawing.

Explore the graphical presentation of the gene

Genbank provides graphical representations of the sequences on its database: click the "Graphics" link below the sequence title, OR click "Display Settings" above the title, and choose "Graphics". Take a few minutes to explore this graphical and compare it with your diagram.

Use BLAST to determine which exons are used in the mRNA transcript

This is the most "bioinformatic" part of the assignment. Blast one of the mRNA sequences (EU076746, EU076747, EU076748, EU076749) against the main sequence (AF527840) and use the results to answer the following questions. Suggested procedures:

  1. Go to the NCBI BLAST website
  2. Click the link “Align two (or more) sequences using BLAST (bl2seq)” under “Specialized BLAST” (near the page bottom)
  3. In the “Sequence 1” text box, type “AF527840” (the accession for the genomics). Fill in “from 1” and “to 34088”. In the “Sequence 2” text box, type in "EU076748" (or other cDNA accession in the table). Fill in the “from” box with 1 and the “to” box with 505.
  4. Click “Align”. You should get a “Blast Result” output page.
  5. Interpret your results:
    1. Which exons are present and which ones are absent in EU076746, EU076747, EU076748, EU076749? (Hint: Refer to the mRNA join statement).
    2. Explain the following BLAST terms: “Expect” (e-value), “Identities”, “Gap”, “Strand”
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