Hybridization Correct Answer 2 ssDNA molecules of comp
base sequence can form a ds hybrid (duplex)
What does the incubation step in hybridization do? Correct
Answer Allows formation of ds molecules
Blocking DNA (Hybridization) Correct Answer minimizes
probe binding to nonspecific sequence
ie salmon sperm DNA, Human LINE-1
Blocking Proteins (Hybridization) Correct Answer minimize
nonspecific binding of probe to membrane
ie casein (milk), Denhardt's sol
Stringency Correct Answer conditions of hybridization that
control the specificity of binding of the probe to the target
sequence
How can you increase strigency in a hybridization?
Correct Answer decrease [salt]
increase [formamide]
increase temp
,Formamide acts as a __________ in a hybridization.
Correct Answer denaturing agent
Line Probe Assay (LiPA) Correct Answer reverse
hybridization assay using sequence-specific
oligonucleotide probes (reverse SSOP)
multi-parameter testing --> single strip
Line Probe Assay steps Correct Answer 1. Isolate nucleic
acid (RNA)
2. Amplify
3. Hybridization
4. Strigent wash
5. Incubate with conjugate
6. Incubate with substrate
7. Detect
What method would you use if you knew the gene
sequence and the mutation? Correct Answer Reverse Dot
Blot
Microarrays Correct Answer Used for unknown gene and
mutation
cDNA libraries
can be used for gene expression, tumors, genetic
mapping, mutations and polymorphism
large scale, high throughput analysis
Microarray steps Correct Answer 1. isolate mRNA from
cells
,2. RT to get labeled cDNA copies of mRNA
3. cDNA washed over slide. cDNA sticks to comp
sequence
4. use laser to read fluorescent tags
Southern Blot Correct Answer Detect a large DNA
fragment among many
Target: DNA, probe: DNA
What can Southern Blots be used to detect? Correct
Answer Deletions/insertions
Point mutations
Polymorphisms
Structural rearrangements
Southern Blot steps Correct Answer 1. RE digest to
fragment DNA
2. Run on gel to separate
3. Soak gel in alkali/NaOH to denature dsDNA
4. Transfer ssDNA fragments to positively charged
membrane (blot)
5. Fix to filter by heat (80C) or UV crosslink
6. Incubate (hybridize) blot w/ radioactively labeled ssDNA
comp probe
7. Autoradiograph
Heterduplex Analysis (HA) Correct Answer use for known
gene, unknown mutation
mutation screening
bands on gel --> retarded migration from WT due to seq
differences
, Heterduplex Analysis steps Correct Answer 1. PCR
2. Mix sample and CTR DNA together
3. Denature PCR using heat
4. Cool slowly to rt
5. Add denaturing loading buffer
6. Run on MDE gel
Single-Stranded Conformational Polymorphism Ananlysis
(SSCP) Correct Answer Used for known gene, unknown
mut
Mutation screening
Short PCR products form 3D conformation when cooled --
> muts have different conformation than WT
Non-denaturing PAGE, muts migrate different than WT
What makes DNA negatively charged? Correct Answer
Phosphate groups of the phosphate:ribose backbone
How does EtBr cause DNA to fluoresce? Correct Answer
Intercalates into the double helix
Absorbs UV ~300 nm, emits ~600 nm
TAE Buffer Correct Answer tris-acetate w/ EDTA
good for DNA recovery
good for lrg fragments
low buffering capacity
increases migration of DNA thru gel
TBE Buffer Correct Answer tris-borate w/ EDTA
good for small DNA fragments
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