- Slides: 18
USING DSAP Step by Step Guide
Using DSAP ■ Go to https: //benzer. rutgers. edu/DSAP/Projects/DSAP 18/login. php (DSAP homepage) and sign in. ■ In order to use DSAP, you need to download a waveform software to be able to analyze the DNA sequences For Mac use: http: //4 peaks. en. softonic. com/mac For PC use: http: //downloads. informer. com/finchtv/download/
Practice Clones (PC 1 – PC 4) ■ In order to get your own real DNA sequence from Rutgers University, they want you to practice using the analyzing software first in order to be ready with your actual DNA sequence. – If you mess up on the actual sequence, it’s money wasted and samples destroyed. ■ You can do the Example Clone (EXC), but that one won’t count towards your practice times.
Opening your Sequence ■ After you have chosen the PC you want to do (1 -4), click on “Welcome” on the left hand side. ■ Click on “Download For Sequence” (This is your DNA Sequences you will be analyzing) ■ Click “Start now” once you opened your sequence.
Crop Ends – For Sequence “ 1. Is this sequence readable? ” ■ You know if the sequence is readable by looking at the sequence and seeing if the peaks are nice and distinguishable (good) or if the peaks are scrambled and mixed together (bad).
Crop Ends – For Sequence “ 2. In the unedited sequence, at which base does the c. DNA insert start? (e. g, C 14)? ” ■ You’re looking for the start of your reading frame. You want to find those Restriction Enzyme sites we discussed on the first lab. ■ Find Eco. RI (AATTC) and right after should be Sfi. IA (GGCCATTACGGCCGGG+) – Look for 3+ Guanines (G) together; the letter after the LAST G is the beginning of the c. DNA Insert.
Crop Ends – For Sequence “ 3. In the unedited sequence, which is the last base of the c. DNA insert or the last base of readable sequence? (e. g. , A 530)” ■ To find where your Clone Insert ends, look for the Poly-A Tail (AAAAAAAA+) – The nucleotide BEFORE the first A of the Poly-A tail is your end letter. ■ If the sequence is unreadable by the time it gets to the Poly-A Tail, then your “stop” is where it begins to be unreadable.
Editing Sequence ■ When editing sequences, you’re taking the ”N” and replacing them with the appropriate nucleotides based on the color pattern shown. – For 4 Peaks: Green = Adenosine (A) Blue = Cytosine (C) Red = Thymine (T) Black = Guanine (G)
BLASTN – SORTING NUCLEOTIDES ■ Click on ”Retrieve Sequence” and copy your sequence from the previous step ■ Click on the link highlighted “BLASTn” and paste your sequence onto the empty box on the top. ■ Push “BLAST” at the very bottom to search the database.
BLASTn - Results ■ You want the results to be in the RED = accurate match, the sequence codes for something PINK = partially accurate, acceptable GREEN = possibly acceptable, try reanalyzing ”Start” and “Stop”” BLUE/BLACK = inaccurate, reanalyze ”Start” and ”Stop” of sequence ■ 1 -1000 bar represents the length of the insert that matched (ex 200 -800 means that at bp 200 to bp 800 matched a function)
BLASTn – “ 2. List the best matches from 3 different organism” ■ You’re using the data from BLASTn to find the 3 organisms that match closest to the nucleotide sequence your insert provided. – Do not use “PREDICTED” or “HYPTHESIZED” results because they are not confirmed results. – Do not use the same organism twice; sometimes databases will show the same organism for a slightly different sequence – **ONLY USE THESE IF NO OTHER OPTIONS POSSIBLE**
BLASTn – est Database ■ BLASTn - est Database is more refined search to help look at the nucleotides in more specific detail.
BLASTx – Searches for Protein ■ BLASTx uses the insert sequence to find a protein that has a similar sequence. ■ Same as BLASTn; Retrieve your sequence and paste it into the sequence box. ■ Push “BLAST” at the bottom to run the sequence
TOOLBOX ■ Toolbox is a software used to help identify the identity of the protein sequence of the insert. ■ Retrieve your sequence like before and click on “Toolbox. ” ■ A new window will pop up and you will paste the sequence into the box
■ Based on the sequence, you will click on ”frame 1, ” “frame 2, ” “frame 3” to see which sequence matches closest to your “start” and “stop” codons. TOOLBOX frames ■ Look for “M” these represent the “START CODON” (methionine – Think Protein Synthesis) ■ Look for “*” these represent the “STOP CODON” ■ UNDERLINE usually means it’s part of your DNA sequence.
Define ORF – 2 and 3 ■ 2. Pick the “Frame” that best meets your DNA sequence ■ 3. Take that DNA sequence from your chosen frame and paste it into the empty box.
Define ORF – 4 and 5 ■ “ 4) At which base does the ORF Start” Use the DNA Sequence that is provided on the chosen Frame to get your Start letter. ■ ” 5) At which base doe the ORF Stop” Use the DNA Sequence that is provided on the chosen Frame to get your Stop letter. (the * represents STOP codon).