Primer Designing No non-specific binding Melting temperature Should not be forming dimers with itself or other primers.
Some thoughts 1. primers should be 17-28 bases in length; 2. base composition should be 50-60% (G+C); 3. primers should end (3) in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming; 4. Tms between 55-80oC are preferred; 5. 3-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; 6. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided; 7. runs of three or more Cs or Gs at the 3-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.Adapted from: Innis and Gelfand,1991
Restriction Analysis Found in Bacteria and archea. 4 types Type -1: cleavage remote to recognition site (methylase activity) Type-2: cleavage within a specific distance Type-3: Cleavage within a short distance Type-4: Cleaves modified DNA (methylated) Ref: http://insilico.ehu.es/restriction/long_seq/ http://molbiol-tools.ca/Restriction_endonuclease.htm
Gene Prediction Patterns Frame Consistency Dicodon frequencies PSSMs Coding Potential and Fickett’s statistics Fusion of Information Sensitivity and Specificity Prediction programs Known problems
Genes are all about Patterns – reallife example
Gene Prediction MethodsCommon sets of rules Homology Ab initio methods Compositional information Signal information
Pattern Recognition in GeneFinding atgttggacagactggacgcaagacgtgtggatgaactcgttttggagctgaac aaggctctatacgtacttaatcaagcggggcgtttgtggagcgagt tacttcacaaaaagctagccaatttgggttcaatgcagtgcctgaccgacatggg tatgtattagtaacgtttggaagaagaaactgttgtggttggtgt ttatgcagacaatctacaggtgactgcaacgaattcaactctcgtggacagttttt tcgttgatttacaggacctctcggtaaaggactatgaagaggtg acaaaattcttggggatgcgcatttcttatgcgcctgaaaatgggtatgattatat atcgagaagtgacaacccgggaaatgataaaggataa atggagaggatgctggagacggtcaagacgaccatcacccctgcgcaggcaatgaag ctgtttactgcacccaaagaacctcaagcgaacctggcccgag cacttcatgtacttggtggccatctcggaggcctgcggtggtacttagtcctgaataacg tcgtgccgtacgcgtccgcggatctacgaacggtcctgat agccaaagtggacggcacgcgtgtcgactacctacagcaagctgaggaactggcgca tttcgcgcaatcctgggagcttgaagcgcgcacgaagaacatt We need to study the basic structures of genes first ….!
Gene Structure – Common sets ofrules• Generally true: all long (> 300 bp) orfs in prokaryotic genomes encode genesBut this may not necessarily be true for eukaryotic genomes• Eukaryotic introns begin with GT and end with AG(donor and acceptor sites) – CT(A/G)A(C/T) 20-50 bases upstream of acceptor site.
Gene Structure Each coding region (exon or whole gene) has a fixed translation frame A coding region always sits inside an ORF of same reading frame All exons of a gene are on the same strand. Neighboring exons of a gene could have different reading frames . Exons need to be Frame consistent! GATGGGACGACAGATAAGGTGATAGATGGTAGGCAGCAG 0 3 6 9 12 01
Gene Structure – reading frame consistency Neighboring exons of a gene should be frame-consistent Frame 0 Frame 2 GATGGGACGACAGATAGGTGATTAAGATGGTAGGCCGAGTGGTC 1 16 33 GATGGGACGACAGATAGGTGATTAAGATGGTAGGCCGAGTGGTC 1 16 40 Frame 0 Frame 2 Exon1 (1,16) -> Frame = a = 0 ; i = 1 and j = 16 Case1: Exon2 (33,100): Frame = b = 2; m = 33 and n = 100 Case2: Exon2 (40,100): Frame = b = 2; m = 40 and n =100 exon1 (i, j) in frame a and exon2 (m, n) in frame b are consistent if b = (m - j - 1 + a) mod 312/7/2012 …31,34,37,40,43,46,49,52…
Codon Frequencies Coding sequences are translated into protein sequences We found the following – the dimer frequency in protein sequences is NOT evenly distributed Organism specific!!!!!!!!!!! The average frequency is ¼% (1/20 * 1/20 = 1/400 = ¼%) Some amino acids prefer to be next to each other Some other amino acids prefer to be not next to each other
Dicodon Frequencies Believe it or not – the biased (uneven) dimer frequencies are the foundation of many gene finding programs! Basic idea – if a dimer has lower than average dimer frequency; this means that proteins prefer not to have such dimers in its sequence; Hence if we see a dicodon encoding this dimer, we may want to bet against this dicodon being in a coding region!
Dicodon Frequencies - Examples Relative frequencies of a di-codon in coding versus non-coding frequency of dicodon X (e.g, AAAAAA) in coding region, total number of occurrences of X divided by total number of dicocon occurrences frequency of dicodon X (e.g, AAAAAA) in noncoding region, total number of occurrences of X divided by total number of dicodon occurrences In human genome, frequency of dicodon “AAA AAA” is ~1% in coding region versus ~5% in non-coding region Question: if you see a region with many “AAA AAA”, would you guess it is a coding or non-coding region?
Basic idea of gene finding Most dicodons show bias towards either coding or non-coding regions; only fraction of dicodons is neutral Foundation for coding region identification Regions consisting of dicodons that mostly tend to be in coding regions are probably coding regions; otherwise non-coding regions Dicodon frequencies are key signal used for coding region detection; all gene finding programs use this information
Prediction of Translation Starts Using PSSM Certain nucleotides prefer to be in certain position around start “ATG” and other nucleotides prefer not to be there 75,100, 75 ATG TCTAGAAGATGGCAGTGGCGAAGA A 0,0,0,100 ,0, TCTAGAAAATGACAGTGGCGAAGA T 100,0,100,0,0, 25, 0, 0 ATG TCTAGAAAATGGCAGTAGCGAAGA G 0, 0, 0, 0, 75 ,0, 0, 25 ATG TCTACT A AATGATAGTAGCGAAGA ATG C 0,100,0,0, 25 ,0, 0, 0 ATG A C G T -4 -3 -2 -1 +3 +4 +5 +6 CACC ATG GC TCGA ATG TT The “biased” nucleotide distribution is information! It is a basis for translation start prediction Question: which one is more probable to be a translation start?
Prediction of Translation Starts Mathematical model: Fi (X): frequency of X (A, C, G, T) in position i Score a string by log (Fi (X)/0.25) CACC ATG GC TCGA ATG TT log (58/25) + log (49/25) + log (40/25) + log (6/25) + log (6/25) + log (15/25) + log log (50/25) + log (43/25) + log (39/25) = (15/25) + log (13/25) + log (14/25) = 0.37 + 0.29 + 0.20 + 0.30 + 0.24 + 0.29 -(0.62 + 0.62 + 0.22 + 0.22 + 0.28 + 0.25) = 1.69 = -2.54 The model captures our intuition! A C G T12/7/2012
Evaluation of Gene prediction TP FP TN FN TPRealPredicted • Sensitivity = No. of Correct exons/No. of actual exons(Measurement of False negative) -> How many are discarded by mistake Sn = TP/TP+FN • Specificity = No. of Correct exons/No. of predicted exons(Measurement of False positive) -> How many are included by mistake Sp=TP/TP+FP • CC = Metric for combining both (TP*TN) – (FN*FP)/sqrt( (TP+FN)*(TN+FP)*(TP+FP)*(TN+FN) ) 12/7/2012
Challenges of Gene finder• Alternative splicing• Nested/overlapping genes• Extremely long/short genes• Extremely long introns• Non-canonical introns• Split start codons• UTR introns• Non-ATG triplet as the start codon• Polycistronic genes• Repeats/transposons