) is a bacterium responsible for many hard-to-treat… Show

) is a bacterium responsible for many hard-to-treat… Show more MRSA (Methicillin-resistant Staphylococcus aureus) is a bacterium responsible for many hard-to-treat infections, in particular in hospitals and healthcare facilities. There are several subspecies of MRSA. All their genomes contain a mecA gene which is known to be responsible for the resistance to methicillin and other β-lactam antibiotics (e.g penicillin). The sequence of the mecA gene in the genome of one MRSA subspecies can be found in the database at: http://www.ncbi.nlm.nih.gov/nuccore/NC_002952.2?report=fasta&from=44919&to=46925&strand=true. As a general practice, treating physicians usually take a swab of the nostrils or sputa and culture the bacteria for confirmatory test. This could take 1-3 days. However, within this period, the bacteria may take hold and develop resistance to treatment. You are asked to develop a methods for the rapid identification of MRSA (<1 hour). One possible method is real-time PCR (using TaqMan probes or SYBR Green). Another method is by PCR followed by gel electrophoresis to identify the length of the fragment. Problems: a. Describe/illustrate the basic principle of PCR (the 3-step cycle and what takes place in each step). b. Describe and illustrate the basic procedure and specify the sequences of a pair of suitable primers for real-time PCR to be performed using cycles consisting of: 1) denaturation at 98 °C --> 2) hybridization at 60 °C –> 3) Synthesis/DNA replication at 72 °C. Hint: 1) Reaction conditions: 1 µM oligonucleotide primers; 5 mM Mg 2+ , 1 mM dNTPs, 50 mM NaCl. 2) You may calculate the Tm of the chosen sequences using a free program based on the nearest neighbor method at this link: http://www.idtdna.com/calc/analyzer. c. Blast your primer sequences to see if they may hybridize to any other sequences available in the databases: https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHomeNew; https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome • Show less

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