1. Computational Analysis of your cDNA Sequences using the
WSSP-14 Chapter 5
Computational Analysis of your
cDNA Sequences using the
DNA Sequence Analysis Program (DSAP)
atttaccgtg ttggattgaa attatcttgc atgagccagc tgatgagtat gatacagttt tccgtattaa taacgaacgg ccggaaatag gatcccgatc atgattgctt caatattttc acttcaatga ttggttctaa gcattcgaat gcgtacccgt ttgattaata tttccatttc tgtcccagtt tttaattttc atttcttttg gttaaaaaat tcccagtctc ttgaatgctt ttctaaaatc tttaattcaa ttatttatta gaatcttctg ttttgagaac tttgtaatgt aattaaataa tttgatgaaa tgattatgaa tgcgaataaa ttattaattt accgtgctga ttggattgaa attatcttgc atgagccagc tgatgagtat gatacagttt tccgtattaa taacgaacgg ccggaaatag gatcccgatc atgattgctt caatattttc acttcaatga ttggttctaa gcattcgaat gcgtacccgt ttgattaata tttccatttc tgtcccagtt tttaattttc atttcttttg gttaaaaaat tcccagtctc ttgaatgctt ttctaaaatc tttaattcaa ttatttatta gaatcttctg ttttgagaac tttgtaatgt aattaaataa tttgatgaaa tgattatgaa tgcgaataaa ttattaattt accgtgttgg attgaaggta attatcttgc atgagccagc tgatgagtat gatacagttt

2. What do I do with this?

3. 4-3

4. 2. Connect to the WSSP website (http://wssp. rutgers
2. Connect to the WSSP website ( and link to DSAP in the Home or Current Project pages
Home Page
Current Project Page
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5. All Teachers must register as a student Different username/email
Log into DSAP
All Teachers must register as a student
Different username/
(Register if first time user)
p. 5-4
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6. My Project Home page - lists clones being worked on
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7. EX1, EX2 – Example clones used in notes
Start the analysis of
an unknown clone
My Clones page
Start the analysis of a clone
EX1, EX2 – Example clones used in notes
PC1-3 – Practice clones, students must complete before the analysis of their unknown clones
PCE4 & 5- Additional Practice Clones Assigned by teacher
Unknown – Clones you have prepped and sequenced
p. 5-6
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8. Notepad - for taking notes on clone
My Clones table:
Notepad - for taking notes on clone
Clone Discussion - for communicating questions on the clone with teacher and staff on a specific clone
p. 5-7
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9. Clone Table: Clone status
Being worked on by a student:
Completed, waiting for review by staff:
Reviewed, needs to be reanalyzed by student:
Corrected by student, waiting for review:
Reviewed and Correct: (PC clones only)
Submitted to NCBI:
Unreadable:
No overlap:
Unable to submit sequence to NCBI:
p. 5-7
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10. Unreadable - Bad sequence Non-coding - Does not code for protein
Clone Table: Remark
Unreadable - Bad sequence
Non-coding - Does not code for protein
Coding - Codes for a protein
Mitochondrial - Mitochondria gene
rRNA - Ribosomal non-protein coding RNA
p. 5-8
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11. Contact – An email link for General, Non-clone specific messages
WSSP Web Pages
DSAP Pages
Links to to
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12. Help and FAQ Links

13. Clone Welcome Page Help Link Discussion Link Navigation Bar p. 5-10
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14. Links to Download Waveform and Programs
Select to download waveform
Links to waveform viewing programs
Go to Next page
p. 5-10
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15. DSAP Crop Ends Q1: Sequence Quality
EX1.14-For waveform
p. 5-11
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16. EX1.14 Waveform
4Peaks
FinchTV
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17. Is this sequence good?
A) Yes B) No
p. 5-9
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18. Are these sequences good?
p. 5-12
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19. Examine Waveform further out
p. 5-13
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20. If the sequence is bad:
Go to next page
p. 5-13
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21. Poor sequence (cont.): Answer question on the Analyze page
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22. Common Causes for Sequencing Problems
Wrong or Bad Primer
Dead Sequencing chemistry (bad reagents)
Blocked capillary (to separate fragments)
Bad Template
Poor Quality
Low Yield
Mixed Templates
© 2013 WSSP

23. Poor Quality Template DNA
Most often caused by ethanol in the plasmid miniprep
(Student forgot to do the second spin after the wash step)
Or not doing the wash step so that SDS is eluted with the DNA
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24. *
b) Low Yield
40-80

25. b) Low Yield (cont.)
High background
Small peaks

26. c) Mixed Templates (Two plasmids)#1 #2
vector
insert
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27. Other Sequencing Problems:
1. Quick stop of good sequences
Usually because ran into the polyA tail (short insert)
Poly A
Tail
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28. 2. Sequences after long runs of repeats may be bad
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29. Slipping of strands during DNA synthesis
Length of DNA
5'-TTTTTTTTTTTTTC
3'-AAAAAAAAAAAAAGCCCTGCTCC-5' N
5'-TTTTTTTTTTTTTC
3'-AAAAAAAAAAAAAGCCCTGCTCC-5' T
N+1
5'-TTTTTTTTTTTTTC
3'-AAAAAAAAAAAAAGCCCTGCTCC-5' A
N-1
N-1 N
N+1
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30. Before Poly-A
After Poly-A
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31. Analyze why the sequence is poor quality and what you would do to fix it
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32. If the sequence is bad (cont.): Submit Page
Enter in a short description
p. 5-12
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33. If the sequence is good:
p. 5-14
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34. Sequence of the pTriplEX2 vector downstream of the sequencing primers.
5’-CTCCGAGATCTGGACGAGC-3’ Primer
3’-GAGGCTCTAGACCTGCTCGAAAAAAAAAAAAAGAGCCCTT-5’ Template
5’-CTCCGAGATCTGGACGAGCTTTTTTTTTTTTTCTCGGGAA-3’ Primer
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35. Look for regions on the waveform that match the polylinker
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36. Synthesis of cDNA from mRNA
p. 1-8
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37. The start of the insert for clone EX1.14
Enter the first base and position of the insert into DSAP
p. 5-17
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38. Question #1. Which base is the start of the For insert?
A) T39
B) A47
C) C54
D) C62
E) C68
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39. Question #2. Which base is the start of the For insert?
A) G49
B) C65
C) G70
D) T72
E) A79
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40. Question #3. Which base is the start of the For insert?
A) T25
B) A32
C) C38
D) C47
E) A52
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41. Question #4. Which base is the start of the For insert?
A) A34
B) C48
C) C57
D) A62
E) T65
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42. Question Which is the proper answer for the start or quality of the sequence?
E) Bad Sequence
A) A36
B) C43
C) A58
D) C72
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43. Determining the endpoint of your sequence
Poor quality
sequence
p. 5-18
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44. Synthesis of cDNA from mRNA
p. 1-8
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45. Identify end of the cDNA insert: Look for end of polyA tail
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46. What if I do not find a poly A tail?
Look to see where the sequence becomes unreliable
p. 5-20
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47. Enter the last base of the insert into the Q3 text box
p. 5-20
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48. Question #3 What base is the end of the insert?
B) T761
C) T847
D) T1095
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49. Crop vector from ends of the EX1.12 sequence:
Method I: Use Crop Selection function
Highlight desired bp for insert
Use Crop Selection Function in Edit pull down menu
p. 5-21

50. Crop vector from ends of the EX1.13 sequence: Method II: Delete ends
Highlight 3' end to delete
Use Delete Selection function in Edit pull down menu
p. 5-22

51. Crop vector from ends of the EX1.13 sequence:
Method II: Delete ends (cont.)
Highlight 5' end to delete
Use Delete Selection function in Edit pull down menu
p. 5-22
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52. Enter cropped sequence into Q4 text box
Move onto next page
p. 5-23
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