Principles of RFLP-PFGE

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Questions and Answers

What is the primary purpose of using restriction enzymes in RFLP-PFGE?

To generate a large number of DNA fragments for analysis

To digest chromosomal DNA into fragments of different sizes (correct)

To separate DNA molecules of 20–1,000 kbp in length

To amplify specific DNA sequences

What is DNA sequencing?

A process of analyzing DNA fragments

A process of replicating DNA

A process of synthesizing DNA

A process of determining or identifying the order of nucleotides present in a DNA sequence (correct)

What is the range of DNA molecule sizes that can be separated by Pulse Field Gel Electrophoresis (PFGE)?

10–100 kbp

1–10 kbp

1,000–10,000 kbp

20–1,000 kbp (correct)

What is an advantage of sequence-based molecular epidemiology?

It provides reproducible typing profiles that are highly amenable to standardization Signup and view all the answers

What is a characteristic of Restriction Fragment Length Polymorphism (RFLP) typing?

It has high reliability and co-dominance. Signup and view all the answers

What is the purpose of Southern blotting in RFLP-PFGE?

To transfer DNA fragments to a membrane Signup and view all the answers

What gene can be used for genotyping bacteria?

Both A and B Signup and view all the answers

What type of sequence typing involves analysis of a particular region of a gene?

Single locus sequence typing Signup and view all the answers

What is the typical temperature and duration of the PFGE process?

14°C for 22 h Signup and view all the answers

What is a limitation of RFLP-PFGE analysis?

It has only moderate sensitivity and may not detect minor genetic changes. Signup and view all the answers

What is an advantage of Single Locus Sequence Typing?

It is a relatively simple and inexpensive method Signup and view all the answers

What is an application of RFLP-PFGE typing?

Conducting an outbreak investigation. Signup and view all the answers

What is the purpose of cell lysis in the RFLP-PFGE procedure?

To release intact chromosomal DNA Signup and view all the answers

What is the main difference between Single Locus Sequence Typing and Multilocus Sequence Typing?

MLST utilizes a larger portion of the genome Signup and view all the answers

What is a problem associated with RFLP-PFGE analysis?

All of the above. Signup and view all the answers

What is the advantage of RFLP-PFGE compared to other molecular typing methods?

It is a fast and simple molecular tool Signup and view all the answers

What is the type of electrophoresis used in RFLP-PFGE to separate DNA molecules?

Pulse Field Gel Electrophoresis (PFGE) Signup and view all the answers

What recent advancement has enabled Whole Genome Sequencing?

Advancement of high-throughput deep sequencing technologies Signup and view all the answers

What is the purpose of staining the gel with ethidium bromide in RFLP-PFGE?

To visualize DNA fragments Signup and view all the answers

Which of the following is NOT a type of sequence typing?

Pulse Field Gel Electrophoresis Signup and view all the answers

What is an advantage of RFLP-PFGE typing over other molecular typing methods?

It provides a high level of discrimination between strains. Signup and view all the answers

What is a reason why RFLP-PFGE typing may not be suitable for high-throughput processing?

It is time-consuming and technically demanding. Signup and view all the answers

Which of the following is a characteristic of RFLP-PFGE typing that makes it useful for distinguishing between closely related strains?

Co-dominance. Signup and view all the answers

What is a benefit of using RFLP-PFGE typing in food safety investigations?

It can detect bacterial contamination in food. Signup and view all the answers

Study Notes

Principles of Fragment Length Polymorphism (RFLP-PFGE)

Chromosomal DNA is digested with restriction enzymes, resulting in fragments of different sizes that form unique patterns when analyzed by agarose gel electrophoresis (PFGE) and Southern blotting.
Differences in these patterns are referred to as RFLPs.
Six nucleotide cutters such as SmaI, XbaI, and SalI are used to digest DNA, generating relatively few DNA fragments for analysis.
Pulse field gel electrophoresis (PFGE) separates DNA molecules of 20-1,000 kbp in length by periodically changing the direction of the electrical field.

Procedure of RFLP-PFGE

Bacterial cells are embedded in a gel block and lysed to release intact chromosomal DNA.
Restriction endonuclease digestion of chromosomal DNA occurs within the gel block.
The gel block is mounted in an agarose gel and DNA fragments are separated by PFGE at 14°C for 22 hours.

Advantages of RFLP-PFGE

A fast, simple, and accurate molecular tool for profiling and identifying populations.
High reliability due to specific sites generated by known restriction enzymes, with results constant over time and location.
Co-dominance allows for distinguishing between heterozygotes and homozygotes, and is highly locus-specific.
Selective neutrality, where different alleles of a gene confer equal fitness.
Good repeatability in the typing of Salmonella gallinarum.
Effective tool for distinguishing closely related strain subpopulations within Lactobacillus delbrueckii.

Limitations of RFLP-PFGE

Requires large amounts of genomic DNA, making the process time-consuming and technically demanding.
Provides a relatively global chromosomal overview, but has only moderate sensitivity, potentially missing minor genetic changes.
Incomplete digestion can lead to difficulty in interpreting band patterns, and complex profiles can be challenging to interpret.

Applications of RFLP-PFGE

Conducting outbreak investigations and resolving false-positive culture investigations.
Identifying clustered patients with possible epidemiologic links.
Detecting bacterial contamination in food (e.g., milk).

Principles of Sequencing

DNA sequencing determines or identifies the order of nucleotides present in a DNA sequence.

Principles of Typing by Sequencing Analysis

Sequence-based molecular epidemiology offers reproducible typing profiles that are highly amenable to standardization, uniform interpretation, and database cataloging.
Universal sequences can be used to genotype bacteria, including 16S rRNA genes, 16S-23S rRNA gene interspacer region, and heat shock protein genes (e.g., hsp65).
Sequence variation in a specific gene can be resolved by sequencing analysis, allowing for molecular epidemiologic applications.

Types of Sequence Typing

Single Locus Sequence Typing (SLST) involves analysis of a particular region of a gene.
Multilocus Sequence Typing (MLST) utilizes a larger, potentially more representative, portion of the genome.
Whole Genome Sequencing (WGS) involves recent advancements in high-throughput deep sequencing technologies.

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Description

This quiz covers the principles of Fragment Length Polymorphism (RFLP) using Pulse Field Gel Electrophoresis (PFGE). It involves digesting chromosomal DNA with restriction enzymes to form unique patterns for analysis.