Which of the following will cause an enzyme to permanently lose its properties I. Hydrolysis II. Freezing to –20°C III. Dissolvi
2 answers:
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Complete question
- It has to be E. coli because it is positive for gapA
- It can't be Salmonella, because it is negative for the invA marker but still makes people sick
- It has to be used because it is positive for the apeE marker
Answer:
<u>1. It can't be Salmonella, because it is negative for the invA marker but still makes people sick </u>
- A combination of positive results from the multiplex PCR inva, apee and gapa is used to definitively identify Salmonella.
- Pathogenic, or disease causing Salmonella is definitely not present, as inva is required for it to be pathogenic. The test did not detect the inva sequence, thus it is truly negative for this particular pathogen.
<u>2. It has to be E. coli because it is positive for gapA </u>
- E. coli may be present, as gapa was detected- a presumptive positive. However, this may need to be definitively determined through further methods of sample analysis, such as 2D-gel electrophoresis.
- apee may belong to another type of bacteria present within the initial sample, or there may be sample contamination
Explanation:
Polymerase chain reactions, PCRs are a form of nucleic acid amplification testing NAAT that exploit the mechanism of transcription by using a thermostable DNA polymerase. These require a sample of genetic material such as RNA or DNA; specific regions of the gene sequence are targeted for replication by primers. In the presence of these specific gene sequences, the primers make billions of copies of the sequences.
However, if these gene sequences are absent, the primers are not capable of identifying and amplifying the sequence. This reaction is highly specific. Positives obtained have a high chance of being true positives and negatives have a high chance of being true negatives .
Pathogens or infectious agents that are capable of causing disease i.e. making people sick. Both E. coli and Salmonella are genuses of enteric bacteria capable of causing disease via fecal contamination. Common symptoms include
- abdominal cramps
- vomiting
- fever
- diarrhea
This test would include primers for the detection of each sequence: gapa, apee, inva. Salmonella's inva was not detected, thus it is not present.
Further steps may include a 2 D gel electrophoresis- here an electrical current is utilized to separate bands of DNA within the sample. This should correspond with an expected DNA size in base pairs or bp for E.coli- this should be determined by running the sample in the gel with a positive control, containing genetic material for E coli, and a negative control, of purified water to determine contamination.
Answer:
The fragment and plasmid are both cut with the same restriction enzyme.
Explanation:
To insert a piece of DNA in a bacterial plasmid, we need to cut both plasmid and DNA insert from same regions. This is typically done by restriction enzymes or restriction endonuclease. This cutting will open the plasmid (which is circular initially) and produce <u>sticky ends.</u> Here, DNA insert can attach because of similar sequence (see attached figure). In the final step, DNA ligase will glue it in the plasmid and it will become its part. This technique has been extensively used as a DNA recombinant technology. A better representation can be seen in the attached figure where both DNA sequence of interest and plasmid are cut with the same restriction enzyme (shown as scissors) and then ligated with DNA ligase.
