Application to find Eukaryotic Open reading frames. Lab

Introduction Finding ORF in Prokaryotic DNA Finding cds in non-intronic Eukaryotes “genes” Use perl scripts (from assignment or elsewhere). Finding ORF in Intronic eukaryotic genes Compare and contrast results of your program with actual results (gene bank or EBI records) Test an online gene finding

Using your assignment code Open the file: ORF pal gene.fasta Find all open reading frames. (This time you must modify your code to translate each codon you can utilise the convertor_hashtable.txt Compare to file: pal protein sequence.fasta. – Visual inspect the files for start and stops; ORFs.; in which reading frame is the TRUE ORF. Compare your results to the results in the gene bank record. Annotation and Analysis site: Pal genePal gene What conclusion can you draw. Note that the file – ORF pal gene.fasta (has extra bases on either side of the ORF take this into account in your analysis)

Predictive translation effect Exons/intron length Consider the following: We have the mRNA CDS of 60 bp in length (start…stop) Let us assume that the intron1 is: – at the end of codon three (position 9) – the length of the intron is 30bp. Intron 2 occurs at: – the end of codon 10 (position 30) – and is 45 bp in length What is the effect of the translations: on Exon A and Exon B? Exon 9 bp Intron 30 bp Exon 21 bp Intron 45 bp Exon 30 Bp BP… ATG TAA Exon AExon B DNA Strand

Predictive translation effect Exons/intron length Consider the following: We have the mRNA CDS of 60 bp in length (start…stop) Let us assume that the intron1 is: – at the end of codon three (position 9) – the length of the intron is 30bp. Intron 2 occurs at: – at position 29 (at the 3 rd bp of codon 10) – and is 45 bp in length What is the effect of the translations: on Exon A and Exon B? Exon 9 bp Intron 30 bp Exon 20 bp Intron 45 bp Exon 31 Bp BP… ATG TAA Exon AExon B DNA Strand

Predictive translation effect Exons/intron length Consider the following: We have the mRNA CDS of 60 bp in length (start…stop) Let us assume that the intron1 is: – at the end of codon three (position 9) – the length of the intron is 30bp. Intron 2 occurs at: – at position 30(the end of codon 10) – and is 43 bp in length What is the effect of the translations: on Exon A and Exon B? Exon 9 bp Intron 30 bp Exon 21 bp Intron 43 bp Exon 30 Bp BP… ATG TAA Exon AExon B DNA Strand

Effect of Translation Example 1 no effect all multiples of 3 Example 2 the last residue of exon 2 is incorrect. The residue for Exon 3 is correct. (but starts at bp 2 of first codon) Example 3 last expn is in different reading frame. Refer to Incorrect_translation_examples.rar

Finding start and stop codons and potential ORF Download the file. (TUBAC3 gene complete sequence) (see last lecture)TUBAC3 gene complete sequence Using “your” perl module and perl script determine the location of the start and stop codons. Write a script that will “attempt” to find at least one ORF. Clearly state how, using your background knowledge of gene expression, you are going to try and resolve this problem. Using your program: determine the position of an (the) ORF(s) for this gene. Discuss how your findings compare with the results obtained in the genebank record file. You can use the graphics image

Intronic DNA gene Go to the understanding Bioinformatics student resources and down load the PRISSEL (CDK10 fasta file). It can also be downloaded from the sample files in the lecture on finding Eukaryotic genes.student resources Run your application to find ORF. Comapre this to the actual results. Discuss your results (in relation to the known experimental results; you can find the actual results in understanding ioinformatics or refer to slide in the lecture: finding Eukaryotic genes.

Online Gene finding program In the student resources you should find links to various online gene predicting applications. Use one to analyse the ALDH10 exon 1 and exon 2 of the ALDH10 gene.