Cytomegalovirus CMV testing Herpes simplex virus HSV testing oAdvantages

Cytomegalovirus cmv testing herpes simplex virus hsv

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Cytomegalovirus (CMV) testing Herpes simplex virus (HSV) testing o Advantages: Quantitative Large number of specimens can be easily tested No special equipment required Total testing time is approximately 3.5 hours Image: Niesters & van Leeuwen, 2019
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51 III. Probe Amplification No change in number of targets (targets not amplified) Synthetic probes (often these are primers) bind to the target sequence, then the probes are copied (not the target) Probe amplification methods: o Ligase chain reaction (LCR) o Strand displacement amplification (SDA) (do not need to know specifics of this one, just that it is a probe amplification method) Ligase Chain Reaction (LCR) o Method for amplifying synthetic probes/primers that are complementary to target sequences o Process: DNA is denatured into single strands Primers are added and anneal to the target sequence In LCR, the primers bind to the target immediately adjacent to each other DNA ligase then glues the primers together to form one strand Ligated primers then serve as the template for annealing & ligating more primers (making copies of primers rather than the target) End-product of LCR = ligated primer strands o Common Uses: Detection of point mutations 1 bp mismatch at the ligation point can prevent ligation Sickle-cell mutation detection C. trachomatis N. gonorrhoeae Image: Ligase Chain Reaction (LCR) presented by Batool, et. Al
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52 Lecture 9, Part II: Hybridization-Based Techniques Objectives Following the lecture, the student will be able to: 1. Define hybridization and describe the process. 2. State the principle for Southern Blot, Northern Blot, & Western Blot tests. 3. Compare Southern Blot and Northern Blot procedures with respect to the target, nucleic acid preparation, probes, and common uses for each. 4. Define the following terms: a. Annealing b. Denaturation c. Renaturation d. Probe e. Target I. Hybridization Technologies Nucleic acid hybridization occurs when two single-stranded nucleic acid molecules (DNA or RNA) with complementary base sequences form a double-stranded hybrid o This technique is used to detect a specific DNA sequence in a mixture of DNA fragments or in total cell DNA Double-stranded DNA can be denatured with heat to form single strands o This is reversible if temperature conditions are right the strands will join back together to form double strands This is known as renaturation Nucleic acid hybridization can occur between: o Single-stranded DNA molecules (DNA:DNA) o Single-stranded RNA molecules (RNA:RNA) o One single-stranded RNA and one single-stranded DNA (RNA:DNA) For hybridization-based techniques, the two populations of nucleic acids must be single- stranded Probes labelled molecules that bind to a specific molecule of interest (target sequence) o Allows us to detect the probe, and therefore detect the target Hybridization-based Molecular Techniques o Southern Blot (DNA) o Northern Blot (RNA) o Western Blot (Proteins, Antibodies)
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53 II. Southern Blot (DNA) Used to detect specific DNA sequence o
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