* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Exercises
Deoxyribozyme wikipedia , lookup
DNA barcoding wikipedia , lookup
Non-coding DNA wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Transposable element wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Frameshift mutation wikipedia , lookup
Genetic code wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Pathogenomics wikipedia , lookup
Genomic library wikipedia , lookup
Human genome wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Point mutation wikipedia , lookup
Metagenomics wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Microsatellite wikipedia , lookup
Smith–Waterman algorithm wikipedia , lookup
Genome editing wikipedia , lookup
Helitron (biology) wikipedia , lookup
Άσκηση 4 Ανάλυση ακολουθιών πυρηνικών οξέων χρησιμοποιώντας το Internet Sequence analysis using Internet functions Short tutorial on restriction mapping, translation, and BLAST. Many of the following exercises involve copying one sequence from a page in Netscape to another. For these types of exercises, therefore, it is a good idea to use multiple windows of Netscape. To create a new window select File - New Web Browser (Ctrl-N). Another method involves the temporary copying of the sequence to an independent window, such as that of a Word document. Restriction mapping. Connect to NCBI Entrez. Retrieve the nucleotide sequence entry with accession code X01405. Mark the sequence from the NCBI page, select Edit-Copy (or Ctrl-C at the keyboard), go to Webcutter (www.medkem.gu.se/cutter) and select Edit-Paste (or Ctrl-V at the keyboard) to introduce the sequence in the sequence window of Webcutter. Click on the button "Analyze sequence" to see restriction sites. We now want to find out where BamHI cleaves. Go back to the main page of Webcutter and deselect "Map of restriction sites". Select "Only the following enzymes" and click on BamHI. "Analyze sequence" gives you the BamHI cleavage sites. How many times does BamHI cleave in the sequence X01405? Go back to the main Webcutter page and reselect "All enzymes in the database". To see all enzymes that cleave exactly once in the sequence select "Enzymes cutting once". The result should be a table where the top looks like: Enzyme Sequence ________ Positions of Recognition Sites Recognition ______________________________________ ___________ AatI AccI AccIII AflIII AlwNI AocI 740 827 1762 408 423 369 agg/cct gt/mkac t/ccgga a/crygt cagnnn/ctg cc/tna . . Other exercises on restriction mapping with Webcutter: www.medkem.gu.se/edu/res.html (in Swedish) or www.medkem.gu.se/edu/4/start.html (in English) Translation. Again retrieve the nt entry with accession code X01405. Translate this sequence using the translation function at www.medkem.gu.se/edu/translat.html. Copy the sequence X01405 as in the restriction exercise above to introduce the sequence in the appropriate box of the page www.medkem.gu.se/edu/translat.html. Click on the "Translate" button to see the three forward reading frames. Try to identify the open reading frame corresponding to the p53 sequence. (You can get help from the annotation section on the NCBI page!). When you have identified the correct reading frame (1 out of 3 possible) do the translation again but select the correct frame (under "Translate entire sequence and select reading frame ...") so that you obtain a linear sequence of amino acids at the top of the result page that corresponds to p53. The sequence (at the top of the page) should look like this: KTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDST 50 PPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGN 100 LRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRP 150 ILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELP 200 PGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALEL 250 KDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD Additional introductory exercises with translation, homology searching and multiple sequence alignment are available at www.medkem.gu.se/edu/3/start.html. Connect to this page and follow the intructions there: Sequence analysis Question # 1 of 8 You have recently been successful with the cloning of a protein gene. You have used the Sanger dideoxynucleotide chain termination method to determine the DNA sequence of one of your clones. What is the sequence read from your sequencing gel ? Analysis of genomic information A few exercises below involves the alignment of 2-4 different sequences. Useful sites are for the alignment of 2 sequences : www-hto.usc.edu/software/seqaln/seqaln-query.html (select global alignment!) genome.eerie.fr/bin/align-guess.cgi Site for multiple sequence alignment : www.medkem.gu.se/ln/molbio/gene/msf.html 1. Alu repeats. Use the SRS or Entrez to retrieve the nucleotide entry Z82206, a sequence from the human chromosome 22 (In SRS select EMBL as database and enter the accession code with the field "AccNumber" . In Entrez specify field as "Accession"). The annotation section may be used to identify Alu repeats ("repeat region .. Alu.."). Part of the feature table of the annotation section will look like: FT FT FT FT FT FT FT FT FT FT FT FT FT FT FT FT FT FT FT repeat_region 1495..1797 /note="AluSp repeat: matches 1..303 of consensus" repeat_region 2171..2375 /note="AluJo repeat: matches 100..302 of consensus" /note="incomplete repeat" repeat_region 2443..2519 /note="MIR repeat: matches 191..260 of consensus" repeat_region 2664..2966 /note="AluSp repeat: matches 1..303 of consensus" repeat_region 3146..4279 /note="TIGGER1 repeat: matches 2418..1298 of consensus" repeat_region 4273..5430 /note="TIGGER1 repeat: matches 1172..1 of consensus" repeat_region 6761..7062 /note="AluSx repeat: matches 1..302 of consensus" repeat_region 7771..8069 /note="AluSc repeat: matches 1..299 of consensus" repeat_region 8289..8692 /note="MLT1A1 repeat: matches 3..365 of consen Use a multiple sequence alignment (www.medkem.gu.se/edu/msf.html) to compare four different Alu repeats. Select for instance the regions 1495..1797, 2171..2375, 6761..7062 and 7771..8069. Are they homologous? 2. Exon - intron structure. a. Identification of a gene with BLAST. Make use of the same sequence Z82206 as above. In the annotation section there is information about an exon (<20814..21617). Use BLAST (www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-blast?Jform=0) to compare this sequence to the database (select program : blastn and database: nr). What seems to be the protein encoded by the exon? b. Alignment of genomic sequence with mRNA. Retrieve the DNA sequences V00594 (Human mRNA for metallothionein) and J00271 (corresponding genomic sequence). Compare these sequences by doing a global sequence alignment. Useful sites are www-hto.usc.edu/software/seqaln/seqaln-query.html or genome.eerie.fr/bin/align-guess.cgi If you are using www-hto.usc.edu/software/seqaln/seqaln-query.html please note: 1. Select Global alignment 2. Enter the two sequences in the 2nd and 3rd larger frames. The first frame is not for sequences! Based on the alignment, how many exons are there in this gene? Compare your result to what's in the annotation section for J00271. 3. Pseudogenes. Consider the following human beta-tubulin sequences: Normal genomic sequence : DNA A processed pseudogene : DNA Normal beta-tubulin : protein Try to examine these sequences to find out why the pseudogene does not give rise to a functional protein. Hints: Translate the pseudogene in all three forward reading frames with the web translation utility (www.medkem.gu.se/edu/translat.html). Compare each of the translation products to the betatubulin sequence (betatub). Once you have identified the relevant reading frame, examine the amino acid alignment between the pseudogene product and the normal beta-tubulin (Make use of www.medkem.gu.se/edu/msf.html). What is the major discrepancy ? What are the significant differences at the nucleotide level?