MSc Medical Biotechnology/ Medical Microbiology “Dry” lab exercises 1. Bioinformatics: 1st exercise In this exercise, we will use online tools to calculate the DNA fragment lengths that would result from digesting bacteriophage DNA with restriction enzymes. You are already familiar with the first part of this from the BT3004 lab last year, but everything is else is new. Navigate to EBI website: http://www.ebi.ac.uk/index.html Choose Nucleotide Sequences. To find the bacteriophage lambda ()sequence: enter the accession number J02459 into the EMBL-fetch box on the left-hand menu. This will bring up the database entry for the sequence. Copy the DNA sequence from the bottom of the page (it’s a very long page). It is about 48000 bases. Navigate to RestrictionMapper: http://www.restrictionmapper.org/ Paste the sequence into the box, and carry out the restriction analysis with enzymes EcoRI and HindIII. Calculate what length fragments you would get if you digest DNA with: EcoRI HindIII EcoRI plus HindIII Put these numbers into a table, and save it (you can do the calculations and make the table using Excel). N.B. Is a linear or circular molecule? This makes a difference to how you interpret the results. 2. DNA Gel electrophoresis (this exercise to be done in your own time, after the computer class). 1 2 3 4 5 6 7 8 The first lane on the left (1, the marker lane) is DNA cut with HindIII plus EcoRI, mixed with D cut with just HindIII. The actual lengths of these fragments are what you calculated from the first exercise. You will need graph paper for this exercise. You can download log graph paper from this site: http://www.saintmarys.edu/~cpeltier/graphpaper/ (i) Plot the distances migrated by each marker fragment, against the log10 of the fragment length. Note the following: Agarose gels like this do not resolve (separate) fragments of DNA >20kb. Fragments that look brighter than they should be, might be 2 coincident or very close bands. The smallest visible marker fragment is 564bp. (ii) Having plotted the calibration curve, estimate the sizes of the DNA fragments in lanes 2 and 4. 3. Bioinformatics: 2nd exercise Imagine you have started your lab project. You have been asked to find a homologue (gene with a common evolutionary origin) for a gene from E. coli in another species of bacteria, Salmonella enterica, and carry out a PCR amplification of it. Here is part of the DNA sequence of the E. coli gene: ATGATCCCGGAAAAGCGAATTATACGGCGCATTCAGTCTGGCGGTTGTGCTATCCATTGCCAGGATTGC In order to find the homologues in other species of bacteria, use the BLAST search. This will compare your query sequence to all other sequences in the database, and find those that are identical or very similar to it. Navigate to this webpage: http://www.ncbi.nlm.nih.gov/BLAST/ Choose Nucleotide BLAST (blastn) and copy and paste the DNA sequence into the search box, select the nucleotide collection (nr/nt) database, then click BLAST. When the results appear, look at the list of matches. Note the species that have homologues of this gene. Find the one for the species you want (S. enterica, strain CT18) by scrolling down the page, where your query sequence has been lined up with each of the matching sequences. Note the nucleotide number at the start of the line labelled “sbjct” (this tells you where in the S. enterica genome the matching sequence is located), and obtain the genomic DNA sequence by clicking on the link to the accession number (blue font, starts with “gi”). Find the CDS (coding sequence, i.e. gene) that corresponds to the matching region of the genomic sequence. Click the blue “CDS” link to obtain the DNA sequence of the gene. (i) What is the function of the protein that the gene codes for? Follow the links and see what you can find out. In order to choose PCR primers for analysis of this gene, open a new browser window and navigate to: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Copy and paste the DNA sequence of the S. enterica gene into the box at the top (the spaces and numbers will be ignored, so don’t bother to edit them out). There are many options that can be changed before the sequence is analysed. The only one we want to change, is to make sure that our PCR product will contain at least bases 100 – 750 of the gene. Use the “Targets” option on the page to set this and then click “Pick primers” to run the analysis. (ii) Record the sequences of the forward and reverse primers the programme has chosen for you, the size of the PCR product that would result, and note the number of alternative primer pairs it gives.