Hybridization and Arrays
- Define hybridization, stringency, and melting temperature
Hybridization: Technique for detecting one molecule within a mixture of molecules to
prove:
● Presence of that molecule
● How much is there
● And/or its approximate size
Stringency: Describes the combination of conditions under which hybridization between
target and probe takes place
Melting Temperature: temperature at which 50% of the nucleic acid is hybridized to a
complementary strand (half of the strand is ds, half is ss)
- List the 2 categories of hybridization techniques and define the principle of hybridization
Two categories
● Solution phase
○ Real-time PCR
● Solid phase (blotting)
○ Southern blots: DNA
○ Northern blots: RNA
○ Western blots: Proteins
Principle: Single-stranded nucleic acids to form specific double-stranded hybrids
● Probe and target are mixed in conditions that allow for specific complementary
base pairing
● Detection of double-stranded hybrids
- Define, very briefly: probe, target, positive and negative controls with regard to hybridization
Probe: oligonucleotide whose identity/ sequence is known
Target: nucleic acid whose identity or abundance is revealed by hybridization
Positive control: sequences that are
● Complementary to the probe
● Assess assay sensitivity
● Ensure that the probe will hybridize to the target under assay conditions
Negative control: sequences without target that determine
● Specificity
● Contamination
- List and briefly describe the steps of solid phase hybridization
Solid Phase: Steps for Blotting
1. Extract the sample (DNA, RNA, protein)
2. Electrophorese the sample on gel
3. Transfer (blot) the gel onto membrane
, 4. Probe membrane (hybridization)
5. Visualize membrane
- Explain stringency and identify conditions related to high and low stringency
High stringency - Conditions require that probe and target match more perfectly to
remain together
● Mismatches do not remain bound together
● Conditions too high = no probe binding to target
Low stringency - Conditions are not so strict
● Mismatches more easily bind together
● Conditions too low = nonspecific binding to unrelated targets
- Identify the detection methods for solid phase hybridization
Autoradiography
Chemiluminescent
Colorimetric
- State how specificity is determined in solid phase hybridization technique
Nucleic acid probes: length confers specificity
● Long probes (500 - 5000 bp) = ↑ specificity
● Short probes (<500 bp) = less specificity
- Diagram the Southern blot procedure
1. Extract DNA from cells
2. Cut with restriction enzyme
3. Run on gel (usually agarose)
4. Denature DNA with alkali
5. Transfer to membrane
6. Hybridization (add probe)
7. Autoradiograph
- Explain the mechanism of action of restriction enzymes (RE) and describe how RE sites are
mapped on DNA
Restriction enzymes cut genomic DNA (Southern Blot)
Type II Cleavage at specific recognition sites
Restriction mapping is a method used to map an unknown segment of DNA by breaking
it into pieces and then identifying the locations of the breakpoints. This method relies
upon the use of proteins called restriction enzymes, which can cut, or digest, DNA
molecules at short, specific sequences called restriction sites. After a DNA segment has
been digested using a restriction enzyme, the resulting fragments can be examined using
a laboratory method called gel electrophoresis, which is used to separate pieces of DNA
according to their size.
- Define hybridization, stringency, and melting temperature
Hybridization: Technique for detecting one molecule within a mixture of molecules to
prove:
● Presence of that molecule
● How much is there
● And/or its approximate size
Stringency: Describes the combination of conditions under which hybridization between
target and probe takes place
Melting Temperature: temperature at which 50% of the nucleic acid is hybridized to a
complementary strand (half of the strand is ds, half is ss)
- List the 2 categories of hybridization techniques and define the principle of hybridization
Two categories
● Solution phase
○ Real-time PCR
● Solid phase (blotting)
○ Southern blots: DNA
○ Northern blots: RNA
○ Western blots: Proteins
Principle: Single-stranded nucleic acids to form specific double-stranded hybrids
● Probe and target are mixed in conditions that allow for specific complementary
base pairing
● Detection of double-stranded hybrids
- Define, very briefly: probe, target, positive and negative controls with regard to hybridization
Probe: oligonucleotide whose identity/ sequence is known
Target: nucleic acid whose identity or abundance is revealed by hybridization
Positive control: sequences that are
● Complementary to the probe
● Assess assay sensitivity
● Ensure that the probe will hybridize to the target under assay conditions
Negative control: sequences without target that determine
● Specificity
● Contamination
- List and briefly describe the steps of solid phase hybridization
Solid Phase: Steps for Blotting
1. Extract the sample (DNA, RNA, protein)
2. Electrophorese the sample on gel
3. Transfer (blot) the gel onto membrane
, 4. Probe membrane (hybridization)
5. Visualize membrane
- Explain stringency and identify conditions related to high and low stringency
High stringency - Conditions require that probe and target match more perfectly to
remain together
● Mismatches do not remain bound together
● Conditions too high = no probe binding to target
Low stringency - Conditions are not so strict
● Mismatches more easily bind together
● Conditions too low = nonspecific binding to unrelated targets
- Identify the detection methods for solid phase hybridization
Autoradiography
Chemiluminescent
Colorimetric
- State how specificity is determined in solid phase hybridization technique
Nucleic acid probes: length confers specificity
● Long probes (500 - 5000 bp) = ↑ specificity
● Short probes (<500 bp) = less specificity
- Diagram the Southern blot procedure
1. Extract DNA from cells
2. Cut with restriction enzyme
3. Run on gel (usually agarose)
4. Denature DNA with alkali
5. Transfer to membrane
6. Hybridization (add probe)
7. Autoradiograph
- Explain the mechanism of action of restriction enzymes (RE) and describe how RE sites are
mapped on DNA
Restriction enzymes cut genomic DNA (Southern Blot)
Type II Cleavage at specific recognition sites
Restriction mapping is a method used to map an unknown segment of DNA by breaking
it into pieces and then identifying the locations of the breakpoints. This method relies
upon the use of proteins called restriction enzymes, which can cut, or digest, DNA
molecules at short, specific sequences called restriction sites. After a DNA segment has
been digested using a restriction enzyme, the resulting fragments can be examined using
a laboratory method called gel electrophoresis, which is used to separate pieces of DNA
according to their size.
