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Transcript of Bioinformatics
melting temperature between 55 and 75°C
melting temperature not more than 5°C different from each other
annealing temperature 10 to 15°C lower than melting temperature
base composition should be 45-65% (G+C)
uniform distribution of G and C nucleotides
avoid long runs of the same nucleotide
check primers are not self-complementary or complementary to other primer
Primers should end (3′) in a G or C, or CG or GC FACTORS TO CONSIDER WHEN DESIGNING PRIMER http://www.maxanim.com/genetics/PCR/pcr.swf PCR Taq polymerase activity
Universal TA cloning PCR Ingredients
DNA template to be amplified PCR Presentation 1 What we learned so far Presentation 2 Proteins involved:
UmuD Acinetobacter Baumanni developed resistance to antibiotics Gene Sequence Protein Sequences Exploring Bioinformatics Software Gene of Interest Using DNA sequence obtained from P2, Lex A Rec A UmuD pGEM-T Vector Direct cloning of PCR fragment (T-A cloning)
Linearized vector with complementary 3’-T overhangs
3’-A overhangs from Taq DNA polymerase
Selection of recombinants: Ampr, lacZ pGEM-T Vector pGEM-T Vector Extra-chromosomal, closed circular double-stranded bacterial DNA
Multiple cloning site
Genes for selection
Exist in high copies Plasmids DNA molecule
Carries DNA to be cloned into host cell
Several important types:
Cosmids Cloning Vector pGEM-T Vector NEBcutter - RecA NEBcutter - RecA Emboss NEBCutter V2.0 Algosome RestrictionMapper Algosome RestrictionMapper Emboss NEBCutter V2.0 Check primers which are given
Things to look out for:
Size of PCR product
Self complementarity score
Self 3' complementarity Getting the Primer
Set the parameters:
Minimum and maximum length of PCR product
Minimum, optimum and maximum annealing temperature
Specificity check against a reference sequence Steps of designing the primer Melting temperature Tm
Secondary structure: No potential to form hairpin loops and no dimerization
No large GC rich or GC deficient areas Criteria for Primers Primer-Blast Obtain the sequence (refSeq or FASTA)
Enter the sequence into Primer Blast Due to the reversed reading of Acinobacter Baumannii genes, the reverse complement of RecA and UmuD PCR products need to be identified.
http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html Reverse Complement of RecA and UmuD Utilization of Sixpack from EMBOSS to translate primer, promoter and mRNA sequence. Checking accuracy of translational products in Silico Compare translated sequence with existing sequences in databases (eg. BLAST) for accuracy. Checking accuracy of translational products in Silico Conclusion Precautions when using Bioinformatics Tool Introducing... Obtaining gene sequence Identifying RE sites Primer Design PCR Vector Selection ORF Proofreading Two primers DNA polymerase with a temperature optimum at around 70 °C Deoxynucleoside triphosphates (dNTPs) Buffer solution Divalent cations, magnesium Monovalent cation potassium ions