DNA Microarrays and Hybridization Techniques

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Questions and Answers

What is the advantage of synthesizing shorter oligos on the chip instead of longer oligos?

It minimizes the need for specialized reagents.

It is much easier and cost-effective. (correct)

It allows for more accurate sequencing.

It reduces the complexity of the design process.

Which step is crucial for ensuring that only one nucleotide is added at a time during oligonucleotide synthesis?

The use of a computer program to control the chip.

The use of a protected nucleotide solution.

The application of multiple nucleotide solutions simultaneously.

The removal of 5'-blocking groups by light. (correct)

During the photolithography process for creating a DNA microarray, what initiates the activation of specific parts of the chip?

Washing with solutions of activated nucleotides.

Exposure to flashing light. (correct)

Application of a solution containing protected nucleotides.

Use of a computer program to determine activation.

What is the first step in preparing for a microarray experiment?

Load probes corresponding to all genes onto a chip. Signup and view all the answers

What is the role of the 5'-blocking groups in the nucleotide synthesis on microarrays?

To prevent unnecessary nucleotide addition. Signup and view all the answers

What type of signal detection occurs after mixing two labeled cDNA pools in a microarray experiment?

A mixture of Red, Green, or shades thereof is analyzed. Signup and view all the answers

What happens during step 3 of the microarray experiment process?

cDNA is labeled with a nucleotide containing a Red fluorescent tag. Signup and view all the answers

What is primarily controlled by a computer during the DNA microarray chip creation?

The primer/oligonucleotide sequences for synthesis. Signup and view all the answers

Which factor does NOT affect the melting point (Tm) of DNA?

Temperature fluctuations Signup and view all the answers

What is the purpose of calculating the melting point (Tm) of DNA in microarray analysis?

To ensure appropriate DNA denaturation Signup and view all the answers

What is the correct formula for calculating Tm of probes that are 14 to 70 base pairs long?

Tm = 81.5 + 16.6(log([Na+]) + 0.41*(%GC) – 600/length) Signup and view all the answers

When given a DNA fragment with 40% GC content and a length of 30 base pairs in a 0.10 M NaCl solution, which component is critical for calculating its melting point?

Molar sodium concentration Signup and view all the answers

Which of the following statements about DNA microarray technology is true?

Microarrays facilitate simultaneous analysis of gene expression. Signup and view all the answers

Which method is NOT provided as an alternative for calculating Tm?

Algorithm based on standard deviation Signup and view all the answers

During a microarray experiment, which consequence is NOT expected if the melting point of DNA is improperly calculated?

Successful specific binding of oligonucleotides Signup and view all the answers

How does the length of a DNA sequence influence its melting point?

Longer sequences generally have higher melting points. Signup and view all the answers

What is one key characteristic of cDNA-based microarrays?

cDNAs are obtained for all genes of interest. Signup and view all the answers

How can the expression levels of genes be inferred from the microarray technology?

By observing the brightness of each glowing spot on the array. Signup and view all the answers

Which type of microarray is likely to be more specific for smaller genomic sequences?

Oligo-based microarrays. Signup and view all the answers

What role do fluorescently labeled nucleic acid probes play in microarrays?

They hybridize to specific gene sequences to enable detection. Signup and view all the answers

What is the main advantage of using microarrays in gene expression studies?

They can examine the expression of thousands of genes simultaneously. Signup and view all the answers

What is an essential requirement for hybridization in microarray technology?

Presence of complementary DNA or RNA sequences. Signup and view all the answers

Which process is primarily used in the manufacturing of oligo-based microarrays?

Photolithography techniques for synthesis. Signup and view all the answers

Which of the following statements about the detection of gene expression via microarrays is true?

Gene expression quantification can be impacted by the quality of sample preparation. Signup and view all the answers

Which factor does NOT influence the melting point (Tm) when calculating for DNA in an aqueous buffer solution?

Presence of cDNA Signup and view all the answers

When calculating Tm using the provided method, which term accounts for the ionic strength of the solution?

$[Na+]$ Signup and view all the answers

In the formula for calculating Tm of probes, what does the constant 600 represent?

A standard adjustment for sequence length Signup and view all the answers

Which of the following conditions would most likely lead to an incorrect Tm calculation?

Using a sequence longer than 70 base pairs Signup and view all the answers

Which method is recommended for students to calculate Tm for assignments and tests according to established guidelines?

The formula provided by Sambrook and Russell Signup and view all the answers

What is the purpose of photoactive blocking groups in the photolithography process of creating a DNA microarray?

They prevent the addition of more than one nucleotide at a time. Signup and view all the answers

In the synthesis of oligonucleotides on a chip, what occurs after specific parts of the chip are activated by light?

A solution with a protected nucleotide is applied to the chip. Signup and view all the answers

During the photolithography process, what is the function of the 5'-blocking groups on the nucleotides?

They limit the reactions to the addition of a single nucleotide at a time. Signup and view all the answers

What is the significance of using a computer during the photolithography process for DNA microarrays?

It programs the desired sequences of nucleotides to be synthesized. Signup and view all the answers

What happens at each spot on the chip during successive washes in the oligonucleotide synthesis process?

Nucleotides are added based on their reactivity to the activated sites. Signup and view all the answers

In the context of oligonucleotide synthesis, what is the result of illuminating the chip with light after adding a nucleotide?

It initiates the deactivation of the 5'-blocking groups. Signup and view all the answers

What is the role of the solution containing one protected nucleotide during the oligonucleotide synthesis process?

To selectively attach a specific nucleotide to the activated site. Signup and view all the answers

What best describes the purpose of photolithography in the creation of DNA microarrays?

To create a sequence-specific pattern on the DNA chip. Signup and view all the answers

Which of the following statements regarding photolithography and its materials used in DNA microarray production is accurate?

Photolithography relies on light-sensitive chemicals to create patterns. Signup and view all the answers

What is the primary advantage of using photolithography in oligo-based microarrays compared to traditional printing methods?

Photolithography enhances the accuracy of nucleotide sequence placement. Signup and view all the answers

In creating a DNA microarray using photolithography, what is the primary role of the activation of specific regions of the chip?

To allow for the selective attachment of nucleotides. Signup and view all the answers

During the process of photolithography in microarray production, which component is crucial for defining the spatial arrangement of oligonucleotides?

The wavelength of the light used for exposure. Signup and view all the answers

Which of the following challenges is commonly associated with photolithography in DNA microarray fabrication?

Poor accuracy in spatial resolution of probes. Signup and view all the answers

Why is photolithography considered a versatile technique for the synthesis of DNA microarrays?

Multiple rounds of synthesis can be performed without changing the substrate. Signup and view all the answers

What is the most significant drawback of using photolithography over other methods in microarray preparation?

It requires expensive and complex equipment. Signup and view all the answers

In the context of photolithography for DNA microarrays, what function do blocking groups serve?

To protect the active sites from unwanted reactions. Signup and view all the answers

Study Notes

DNA Microarrays

Microarrays use oligonucleotides and cDNA to analyze gene expression.
Study by Barrett and Kawasaki (2003)
Pages 248-250 of the textbook

DNA Denaturation and Renaturation

DNA can be denatured by heat or high pH.
Denaturation separates the DNA strands.
The temperature at which DNA denatures is the melting point (Tm).
Upon removal of the denaturing agent, DNA renatures to a double-stranded form.
Factors affecting Tm include GC/AT content, salt concentration, DNA concentration, DNA length, and modifications.
Tm can be calculated using a formula (Tm = 81.5 + 16.6(log [Na+]) + .41* (%GC) – 600/length).

Calculating Tm

Formula: Tm = 81.5 + 16.6(log [Na+]) + 0.41* (%GC) – 600/length
[Na+] is the molar sodium concentration.
%GC is the GC ratio.
Length is the length of the sequence.

DNA Hybridization

DNA molecules can form hybrids.
The strength of hybridization depends on the degree of homology.
DNA hybridization has diverse applications (e.g., DNA/RNA probes, blotting, microarray experiments, in situ hybridization).

Application of DNA Hybridization: Southern/Northern Blotting

DNA/RNA is resolved on a gel.
DNA/RNA is transferred to a membrane.
A labelled probe is prepared and allowed to bind to the target gene on the membrane.
The signal produced by the probe is detected using a suitable method (e.g., autoradiography).

Detection of PECAM-1 and GAPDH mRNA Levels

Northern blotting was used to study the mRNA levels of PECAM-1 and GAPDH in different tissues.
Results from the figure show the differing levels in different tissues

Large-Scale Gene Expression Studies using Microarrays

Microarrays are used to study large-scale gene expression at the level of transcription.
Microarrays can study the expression of all genes in an organism.
Example: Comparing gene expression in yeast before and after spore formation.
Visualized as an array of spots, each representing a gene.

Types of Microarrays

cDNA-Based Microarrays:
- cDNAs are obtained for genes of interest.
- cDNAs are loaded onto a chip.
- The chip is hybridized with labeled cDNA probes corresponding to a given tissue.
Oligo-Based Microarrays:
- Oligos are synthesized based on sequence information for genes of interest.
- Longer oligos may be synthesized separately and loaded onto the chip.
- Shorter oligos may be synthesized on the chip (more efficient and cost-effective).

Photolithography to Create a DNA Microarray

Probe sequences are directly synthesized on a chip.
A computer program guides the creation of desired sequences.
Reactive groups of nucleotides are initially inactive using photoactive blocking groups that can be removed by a flash of light.
- Specific steps described in the figure provide details on the photolithography process.

Steps involved in a Microarray Experiment

Load probes onto the chip.
Extract mRNA from cells under different conditions.
Label cDNA from mRNA pools with fluorescent tags (e.g., Red and Green).
Mix cDNA pools and allow hybridization.
Scan the array to detect red, green, or blended colors.
Analyze the data to understand gene expression differences.

Microarray Experiment Results

Gene spots producing red or green signals indicate the gene's expression in one condition.
Blended color spots indicate expression in both conditions
Spots with differing shades of blended colors indicate differential expression levels.
Equally expressed genes show yellow signals.

Additional examples

Example 2: Using microarrays to monitor gene expression in a frog at different developmental stages.

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Description

Explore the concepts of DNA microarrays, denaturation, renaturation, and hybridization techniques. This quiz draws from Barrett and Kawasaki's study and covers critical formulae for calculating melting points and factors influencing DNA structure. Test your understanding of gene expression analysis and DNA interactions.