PCR and Southern blotting

What changes can you do when PCR has no target bands

For little to no target band: 

1.     Check your math 

2.     Verify all components are present 

3.     Verify primer design 

4.     Consider if DNA sample has inhibitors (amplified successfully in other reactions?) 

5.     Lower annealing temperatures 

6.     Add more template or cycles 

7.     Add more magnesium 

8.     Use PCR enhancers or higher denaturation temps for GC-rich targets 

What can you do if your PCR has non-specific products

For non-specific products: 

1.     Increase annealing temperature 

2.     Reduce primer concentrations (especially if there are abundant primer dimer complexes) 

3.     Shorten annealing and extension times 

4.     Reduce cycles or template 

5.     Reduce magnesium 

What are the steps to southern blotting

Southern blot steps: 

1. Restriction enzyme digest 

• DNA is isolated and then cut  

2. Electrophoresis of agarose gel  

• Fragments are separated by gel electrophoresis, denatured in the gel 

3. Transfer DNA to nitrocellulose membrane (blotting) 

• Fragments are transferred to a solid support membrane 

4. Hybridize probe to plot 

• DNA fragments on the membrane are exposed to a labeled probe that is complementary to the region of interest 

• Probes are usually larger (allow to see individual bands) 

  • Wash blot 

5. Detection of probe signal 

• The signal of the probe is detected to indicate the presence or absence of the sequence of interest 

What is dot/slot blots

Dot/slot blots: target DNA/RNA is deposited directly on the membrane by means of various devices (ex: vacuum system) 

• Applied to expression, mutation, and amplification/deletion analyses 

  • Determination of size is not required 

• Most efficient on less complex samples 

Dot blots = target is deposited in a circle or dot 

• More useful for multiple qualitative analyses where many targets are being compared (mutational screening) 

• Ability to test and analyze larger numbers of samples at the same time 

Slot blots = target is deposited in an oblong bar 

• More accurate for quantification by densitometry scanning because they eliminate the error that may arise from scanning through a circular target 

What is macroarrays (reverse dot blot)

Macroarrays (reverse dot blot): many different unlabeled probes are immobilized on the membrane, and the test sample is labeled for hybridization with the immobilized probes 

• a known sequence is immobilized at a known location on the blot and the amount of sample that hybridizes to it is determined by the signal from the labeled sample 

• Limited by the area of the membrane and the specimen requirements 

What is microarrays

3. Microarrays:

• Tens of thousands of targets could be screened simultaneously in a very small area by miniaturizing the deposition of droplets 

• Array targets immobilized on glass slide  

  • Targets can be DNA, RNA, or protein 

• Requires fluorescent reader and analysis software 

• Probes are immobilized on a solid support 

What is comparative genome hybridization (CGH)

Comparative genome hybridization (CGH): designed to test DNA 

• Used to screen the genome or specific genomic loci for deletions and amplifications 

• Genomic DNA is isolated, fragmented, and labeled for hybridization on the chip 

• Provides higher resolution and more defined genetic information than traditional cytogenetic analysis 

• Limited to the analysis of loci represented on the array 

• Advantage = can be performed on fixed tissue and limiting samples 

What is bead arrays

Bead arrays: immobilize probes with beads, allowing hybridization of the targets in the bead suspension 

• Used for protein and nucleic acid targets 

• Available for infectious disease and tissue typing 

• Beads are color coded with a particular shade of red fluorescent dye so you can distinguish specific probes carried on different beads 

What are the concepts and applications of the following hybridization method: Dot/slot, macroarrays, microarrays, CGH, and bead arrays

look at pic

What are applications of southern blots

Applications of southern blots: (southern blotting is “old school”) 

• Genetics, oncology (translocations, gene rearrangements) 

• Detection of repeat expansions (FXS, Huntington) 

• Typing/classification of organisms 

• Cloning/verification of cloned DNA 

• Forensic, parentage testing (RFLP, VNTR) 

What is the purpose and probe type of Southern, Northern, and Western blots

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What are factors that affect stringency in blotting

Factors that affect stringency:  

• Temperature of hybridization 

  • High temperatures = higher stringency 

  • Low temperatures = lower stringency 

• Wash temperature

  • high temperatures = higher stringency

  • low temperatures = lower stringency

• Hybridization time

  • more time = lower stringency

  • less time = higher stringency

• Wash time

  • lower time = lower stringency

  • higher time = higher stringency

• Salt concentration of hybridization buffer 

  • Keep hybridization solution low  

  • High salt = lower stringency 

  • Lower salt = higher stringency 

• Concentration of denaturant (formamide) in the buffer 

  • Formamide lowers the optimal hybridization temperature 

  • More formamide = more stringency 

• Length and nature of probe 

  • Long probe or high GC bases = binds in more stringent conditions  

    • Require longer hybridization times 

  • Short probe or high AT bases = binds in lower stringent conditions 

    • Require lower hybridization times 

  • Increased probe concentration = increased sensitivity of analysis 

Ideal conditions = calculated with Tm of probe sequence (and Cot) 

How does stringency relate to probe binding

High stringency = more demanding of probe/target complementarity and length 

• If too high, the probe will not bind to target 

Low stringency = more forgiving binding 

• If too low, the probe will bind to unrelated targets 

How is stringency in PCR and southern blotting similar

look at the pic