Term
| At what temperature range is Double-stranded DNA denatured by heat to form single strands? |
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Definition
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Term
| Denaturation is reversible if the temperature if kept at what temperature? |
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Definition
| 65 degrees Celsius or lower |
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Term
| Reversible Denaturation is also known as? |
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Definition
| DNA renaturation or Hybridization |
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Term
| When DNA is denatured which bonds holding the complementary DNA are broken? |
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Definition
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Term
True or False:
DNA Hybridization can occur between any single stranded Nucleic Acid Chain |
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Definition
|
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Term
| What is the definition of Hybridization? |
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Definition
| Formation of stable double stranded nucleic acid structure due to complementary base pairing and formation of hydrogen bonds |
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Term
| What does Hybridization detect? |
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Definition
|
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Term
| What is the target that Hybridization detects? |
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Definition
| Specific nucleic acid sequence |
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Term
| What is complementary and hybridizes to target sequence? |
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Definition
|
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Term
| What can Hybridization occur between? |
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Definition
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Term
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Definition
| DNA or RNA fragment used to detect specific DNA or RNA in mixture |
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Term
| With the probe what is complementary to region of interest? |
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Definition
| Short Single-stranded Oligonucleotide |
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Term
| Name 4 Labels for detection |
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Definition
Radioactive: 32P
Fluorescent
Chemiluminescent
Enzyme |
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Term
| What does the radioactive label: 32P detect? |
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Definition
| Probe-target hybrid by autoradiography |
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Term
| What is the 2 step process for Radioactive: 32P label? |
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Definition
Expose X-ray film to hybridization reaction
Probe radioactivity alters X-ray film |
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Term
| What is the "Direct detection" method? |
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Definition
Detects presence of "target" nucleic acid sequence
PCR different in that target amplified & probe not needed |
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Term
| Is the probe needed in "Direct detection" method? |
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Definition
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Term
| Name the 4 Hybridization-based Molecular Techniques |
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Definition
Southern Blot: DNA
Northern Blot: RNA
Western Blot
FISH |
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Term
| What does FISH stand for? |
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Definition
| Fluorescent in situ hybridization |
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Term
| What does the Western Blot detect? |
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Definition
| Proteins using antibodies OR Antibodies using Proteins |
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Term
| Is Western Blot a nucleic acid hybridization technique? |
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Definition
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Term
| Hybridization "steps" used in other techniques: DNA Sequencing |
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Definition
| Primers hybridize to DNA to be sequenced |
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Term
| Hybridization "steps" used in other techniques: PCR |
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Definition
| Primers must bind to sequence to be amplified |
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Term
| What does Southern Blotting Detect? |
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Definition
| Specific DNA fragments in complex mixture |
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Term
| What can the target be in Southern Blotting? |
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Definition
| Single gene or larger part of DNA |
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Term
| Who invented Southern Blotting? |
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Definition
| Edward Southern in the 1970s |
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Term
| Which Hybridization-based Molecular Technique was the first to be developed? |
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Definition
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Term
True or False:
Southern Blotting is becoming obsolete with newer faster techniques becoming available (e.g. PCR) |
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Definition
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Term
| List the 7 steps of Southern Blotting Procedure |
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Definition
1. DNA treated with REs
2. DNA fragments separated by electrophoresis
3. Nitrocellulose paper applied to gel and DNA fragments transferred to paper
4. Paper placed in solution with radioactive probe
5. Paper rinsed to remove unattached probe
6. Paper overlaid with X-ray film
7. Film examined for changes indicating position of radioactive probe |
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Term
|
Definition
|
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Term
True or False:
Hybridization does not work well in gel |
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Definition
|
|
Term
True or False:
Nitrocellulose paper process stabilizes fragments so they will not move |
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Definition
|
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Term
| What does the Strong Alkaline Buffer do? |
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Definition
| Denatures Double-Stranded DNA into single strands |
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|
Term
True or False:
DNA must be single-stranded before probe can hybridize |
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Definition
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Term
Southern Blot - Detection of Sickle Cell Mutation
Normal: 1 RFLP band at ____ kb |
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Definition
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|
Term
Southern Blot - Detection of Sickle Cell Mutation
Carrier: 1 RFLP band at ____ kb and 1 RFLP band at ___kb |
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Definition
|
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Term
Southern Blot - Detection of Sickle Cell Mutation
Affected: 1 RFLP band at ____ kb |
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Definition
|
|
Term
Which type of Hybridization technique is the following:
1. DNA extracted and treated with MstII RE
2. MstII cuts DNA at multiple sites --> RFLPs
3. Sickle cell mutation alters one of MstII's sites
4. Record Results |
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Definition
|
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Term
| With Southern Blot is a DNA ladder of known size run with test for reference? |
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Definition
|
|
Term
| What is Northern Blotting used for? |
|
Definition
| mRNA studies - Identify "expressed" genes |
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|
Term
| mRNA is relatively ______ |
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Definition
|
|
Term
| Do you need to cut mRNA with REs? |
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Definition
|
|
Term
| What specifically is Northern Blotting identifying with "expressed" genes? |
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Definition
Genes being transcribed/translated
Expression by abnormal genes |
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Term
| Is the DNA under study with FISH still in original place in cell or on the chromosome rather than being extracted? |
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Definition
|
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Term
| With FISH what are the DNA & Chromosomal proteins fixed with? |
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Definition
|
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Term
| FISH - How do you denature chromosomes to single strands? |
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Definition
|
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Term
| What are the 4 parts of the Principle of FISH? |
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Definition
1. Probe specific for target
2. Labeled with fluorescent dye
3. Probe added to specimen, allowed to anneal, and then washed to remove probe
4. Examine under fluorescent microscope |
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|
Term
| What are the 3 uses of FISH in general |
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Definition
Locus specific
Centromere probe
Spectral Karyotyping Whole Chromosome Paints |
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|
Term
Describe the following:
FISH - Locus Specific |
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Definition
Hybridize to a particular chromosome region
Study gene & its location |
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|
Term
Describe the following:
FISH - Centromere Probe |
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Definition
| Can identify and count number of chromosomes since the centromere nucleotide sequence is unique |
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Term
|
Definition
| Constricted area in chromosome |
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|
Term
Describe the following:
FISH - Special Karyotyping (SKY) whole chromosome paints |
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Definition
*Collections of smaller probes
*Pieces of same chromosome stained with same dye |
|
|
Term
| What is SKY used to detect? |
|
Definition
|
|
Term
| What does Each Piece of SKY do? |
|
Definition
| hybridizes to different sequence along length of same chromosome |
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|
Term
| What is Chronic Myelogenous Leukemia (CML) considered? |
|
Definition
|
|
Term
|
Definition
ABL gene on Chromosome 9 moves near BCR gene on Chrosome 22
-PHILADELPHIA CHROMOSOME |
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Term
| How many probes is CML treated with? |
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Definition
|
|
Term
| What are the microscopic exam results for CML? |
|
Definition
1 ABL gene
1 BCR gene
1 Combination gene |
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|
Term
| What is HER2/neu considered as? |
|
Definition
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