Capillary DNA Analysis

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Questions and Answers

What was the primary use of Northern blots?

Research tool for protein analysis

What is the purpose of using serum or biological fluids as probes in Western blots?

To detect the presence of antibodies produced in response to infection

What was the limitation of Southern blotting and its variations?

It could only assess one or a few molecular targets

What is the primary difference between genomics and traditional molecular analysis?

The number of targets analyzed Signup and view all the answers

What is the purpose of reverse-dot-blot hybridization?

To perform simultaneous analysis of many genes or proteins Signup and view all the answers

What is a characteristic of modern arrays?

They can carry up to hundreds of thousands of probes Signup and view all the answers

What is the advantage of Western blots over other methods?

It is highly specific Signup and view all the answers

What is the primary application of Western blots in research laboratories?

To perform protein analysis Signup and view all the answers

What is the primary purpose of an internal amplification control in qPCR and RT-qPCR?

To confirm that negative results are true negatives Signup and view all the answers

What is the relationship between the amount of target nucleic acid and the Ct value?

Inversely proportional Signup and view all the answers

What is the application of qPCR in detecting microorganisms that are difficult or dangerous to culture?

Detection of viruses and other pathogens Signup and view all the answers

What is the purpose of the standard curve in qPCR?

To convert Ct values to DNA copy numbers Signup and view all the answers

What is the advantage of digital droplet PCR over traditional qPCR?

Absolute quantification Signup and view all the answers

What is the purpose of multiplex qPCR?

To analyze the expression of multiple genes Signup and view all the answers

In RT-qPCR, how is the level of target quantified?

Relative to an internal amplification control Signup and view all the answers

What is the application of qPCR in bone marrow transplantation?

Measurement of donor bone marrow in the recipient Signup and view all the answers

What is the purpose of attaching a fluorescent label to DNA chains in capillary electrophoresis?

To enable detection of the DNA chains by laser excitation Signup and view all the answers

What is the function of the laser inside the capillary instrument?

To excite the fluorescent labels attached to the DNA chains Signup and view all the answers

What is the significance of the molecular weight standard in capillary electrophoresis?

It provides a reference point for estimating the size of the DNA fragments Signup and view all the answers

What is the difference between the agarose gel in traditional electrophoresis and the electropherogram in capillary electrophoresis?

The agarose gel displays bands, while the electropherogram displays peaks Signup and view all the answers

Why is it necessary to denature the DNA into single strands in capillary electrophoresis?

To enable the DNA fragments to migrate through the capillary Signup and view all the answers

What is the purpose of the nucleic acid-specific fluorescent dye in traditional electrophoresis?

To visualize the nucleic acid fragments on the agarose gel Signup and view all the answers

How are the lengths of the nucleic acid fragments determined in capillary electrophoresis?

By comparing their migration patterns to the molecular weight standard Signup and view all the answers

What is the advantage of using capillary electrophoresis over traditional electrophoresis?

All of the above Signup and view all the answers

What is the primary advantage of massive parallel sequencing over other methods?

It enables the analysis of all known genetic loci for clinically significant alterations Signup and view all the answers

What is the significance of tumor mutational burden in cancer treatment?

It affects treatment strategy decisions and disease prognosis Signup and view all the answers

What is the role of bioinformatics in the interpretation of genomic data?

To merge biological data with information technology Signup and view all the answers

What is the requirement for adequate sample DNA in NGS?

To provide sufficient coverage of regions of interest Signup and view all the answers

What is the recommended probability of error for clinical sequencing?

1/1,000 Signup and view all the answers

What is the purpose of targeted gene panels in NGS?

To produce a finished report with variants identified automatically Signup and view all the answers

What is the significance of large databases in genomic analysis?

To facilitate future sequence analysis Signup and view all the answers

What is the challenge of interpreting genomic data generated by NGS?

The requirement for massive storage space Signup and view all the answers

What is the primary function of DNA polymerase in the pyrosequencing reaction?

To form a phosphodiester bond between the primer and the nucleotide Signup and view all the answers

What is the maximum length of sequence information that can be produced by pyrosequencing?

100 bases Signup and view all the answers

What is the purpose of adenosine 5′ phosphosulfate (APS) in the pyrosequencing reaction?

To convert PPi to ATP Signup and view all the answers

What is the advantage of pyrosequencing over Sanger sequencing?

It is less labor intensive Signup and view all the answers

What is the result of incorporating a non-complementary nucleotide in the pyrosequencing reaction?

No signal is produced Signup and view all the answers

What is the purpose of introducing dNTPs sequentially in the pyrosequencing reaction?

To determine the correct base in the sequence Signup and view all the answers

What is the result of the pyrosequencing reaction?

A pyrogram of luminescent peaks is generated Signup and view all the answers

Why is pyrosequencing still in use despite its limitations?

It is useful for targeted sequencing of specific regions Signup and view all the answers

Study Notes

DNA Detection and Analysis

DNA chains must carry a fluorescent label to be detected in capillary instruments
A laser inside the capillary instrument excites the fluorescent labels as they move through the capillary
The dyes emit fluorescence, which is detected and transferred to a computer as an electrical signal

Capillary Electrophoresis

Replaces traditional gel electrophoresis with an electropherogram
Peaks represent the nucleic acid fragments, and the instrument computes and displays the lengths of the nucleic acid fragments in base pairs
Multiple samples can be run simultaneously through a single capillary

Preparation of Capillary Gel Electrophoresis

Involves loading premixed polymer solution and buffers into the electrophoresis instrument along with the capillary
Polymer is automatically injected into the capillary by the instrument
Nucleic acid samples are diluted in formamide to denature the DNA into single strands, and the molecular weight standard is added directly to the sample

Western Blot

Used to detect proteins and protein modifications
Serum, cell lysate, or extracted proteins are separated by gel electrophoresis and blotted to a membrane
Probes for western blot are polyclonal or monoclonal antibodies specific for the proteins of interest

Array Methods

Allow for the simultaneous analysis of many genes or proteins
Modern arrays can carry up to hundreds of thousands of probes
Genomics refers to the analysis of hundreds to thousands of targets or whole genomes

Quantitative PCR (qPCR)

Measures the amount of target nucleic acid in a sample
The fluorescence plotted versus cycle number generates a curve similar to a bacterial growth curve
The length of the lag phase is assessed by counting the number of PCR cycles required to reach a threshold level of fluorescence
The cycle at which the sample fluorescence reaches this value is called the threshold cycle or Ct

qPCR Applications

Detection of microorganisms, especially viruses and other pathogens that are difficult or dangerous to culture in the laboratory
Tumor-associated gene expression
Tissue typing
Multiplex qPCR methods can assess multiple targets simultaneously

Digital Droplet PCR

Relies on the generation of light (luminescence) when nucleotides are added to a growing strand of DNA
No gels, fluorescent dyes, or ddNTPs are required
Procedure is used to measure the donor bone marrow in the recipient after a bone marrow transplant

Pyrosequencing

Generates a pyrogram of luminescent peaks associated with the addition of the complementary nucleotides
Produces short- to moderate-length sequence information (up to 100 bases)
Less labor-intensive than Sanger sequencing

Whole Genome Sequencing

Primarily a research tool
Massive parallel sequencing has the capacity to investigate all known genetic loci for clinically significant alterations
Tumor mutational burden (number of mutations/Mb of sequenced DNA) has been identified as a biomarker for some types of cancer

Bioinformatics

Merges biological data with information technology
The interpretation of data generated by NGS requires massive storage space and contributes to continual renewal of previously stored data in organized databases
Database information can be used to refine interpretation of newly collected data

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