| DNA | - deoxyribonucleic acid
- double helix structure
- consists of nucleotide bases: adenine, cytosine, guanine, and thymine (AT, GC)
- two strands held together by H-bonds
- heat or high pH can separate strands |
| High pH conditions | - lyses bacterial cells to release DNA
- denatures DNA: separation of the double-stranded DNA into single strands |
| DNA probe | - short piece of ssDNA
- hybridizes with a specific nucleotide sequence
- contains radioactive phosphorus or sulfur in its nucleotides
- labeled for detection
Labels -->
Isotopic: 32P, 35S, 125I
Enzymes: alkaline phosphatase, horseradish peroxidase |
| Pathogen detection with a DNA probe | 1. Prepare food homogenate
2. Filter food homogenate
3. Trap cells on nitrocellulose filter
4. Place filter on agar and incubate
5. Apply DNA hybridization technique to detect pathogen |
| DNA hybridization technique | 1. Isolate cells on solid support (ex. filter)
2. Lyse cells
3. Denature DNA
4. Add labeled probe
5. Probe hybridizes to target DNA
6. Check for signal (radioactivity or color) |
| Nucleic acid methods | basic principle: detect specific gene(s) or DNA
DNA probe: single stranded probe specific for a unique genetic sequence of the target organism
- organisms in the food sample fixed to solid support (ex. membrane filter)
- cells lysed and DNA denatured
- labeled DNA probe added to membrane filter
- filter placed on agar surface
- microbial colonies transferred to membrane and blotted with probe
- probe attaches only to complementary sequence
- probe detected by radioactive or enzyme label |
| DNA probe applications | - rapid detection of pathogens
- distinguish virulent from avirulent strains
- identify antibiotic resistance genes |
| DNA probes: pros and cons | advantages:
- specificity for target organism
- sensitivity (can detect low numbers of microbes)
- can get results faster than traditional methods |
| DNA probes: disadvantages | - DNA probes detect viable and non-viable cells
- provide less information than cultural methods
- enrichment step can reduce rapidity |
| Polymerase Chain Reaction (PCR) | - method of amplifying DNA
- makes may copies of a particular DNA sequence
- specific primer sequences used
- can increase sensitivity of DNA probe assays
- multiple cycles of DNA replication result in amplification to 10^7 molecules
- DNA sequence detected via agarose gel electrophoresis and labeling with probe |
| Polymerase Chain Reaction (PCR) materials required | - target DNA for amplification
- primers (20 nucleotides long, provide starting point for DNA synthesis)
- DNA polymerase (Taq polymerase)
- Nucleotides (A, T, G, C)
- Thermocycler |
| DNA Polymerase | - Taq polymerase
- enzyme from Thermus aquaticus
- high thermostability
- high activity
- very low error rate |
| PCR proces | each 3 step process --> one cycle
1. denature target dsDNA to ssDNA
2. anneal (join) primers
3. extend primers by enzymatic addition of nucleotides
- each cycle doubles the amount of DNA target
- typical amplification is 20-40 cycles
- results in a million-fold amplification of DNA |
| PCR step 1 | Denaturation
- reaction mixture heated to 95 C
- dsDNA of target separate into ssDNA
when target is RNA, use reverse transcriptase enzyme to first convert RNA to DNA before heat is applied |
| PCR step 2 | Annealing
- reaction mixture temp reduced to 55 C
- primers anneal to ssDNA
- must have excess primers |
| PCR step 3 | Extension
- reaction mixture heated to 70 C (optimum activity of Taq polymerase)
- Taq polymerase builds complementary strands |
| Detection of Amplified DNA (PCR) | - agarose gel electrophoresis
- labeled DNA probe applied to detect target DNA |
