PCR radiolabeling

With a "good" probe sequence, RadiolabelingDna by random priming is fast and convenient, however, sometimes greater sensitivity is needed. In these cases, generating a radiolabeled probe by PCR can be used. The probe has higher overall specific activity because much less unlabeled DNA template is required for PCR than for random priming. To avoid chain termination by misincorporation of unlabeled dNTPs, it is important to decrease the concentration of the unlabeled dNTPs to be similar to that of the concentration of the limiting radiolabeled dNTP (cold nucleotides each at 2x the concentration of the limiting nucleotide works well).

This protocol works well with probes from about 500 bp to 1000 bp in length.

	PCR reaction
2.0 μl	10x TAQ buffer with MgCl2 (NEB)
2.0 μl	supercoiled plasmid DNA template at 50 pg / μl (100 pg total)
2.0 μl	forward primer at 1 μM
2.0 μl	reverse primer at 1 μM
5.0 μl	α-32P-dATP (50 μCi @ 3000 Ci/mmol)
0.4 μl	TAQ DNA polymerase at 5 U / μl (NEB)
2.0 μl	dGTC-TP at 40 μM each (the other three cold nucleotides)
1.0 μl	dATP at 20 μM (not radioactive → helps the synthesis reaction happen)
3.6 μl	H2O
20 μl	total volume

		typical cycling parameters
3'	94C	initial denaturation
30"	94C	denaturation
30"	55C	anneal (may need to be optimized)
1'	72C	extension, + 2" per cycle
		30 cycles
7'	72C	final extension
	4C	hold

To label DNA molecular weight markers:
Lambda phage (λ) molecular weight markers on a Southern membrane can be lit up by radiolabeling λ genomic DNA. λ digested with BstEII is available from New England Biolabs. The BstEII restriction enzyme leaves 5' overhangs that can be filled in by residual TAQ and nucleotides (either α³²P-dATP or ³²P-dCTP will work) after the PCR radiolabeling reaction is completed.

• after the complete PCR radiolabeling reaction is finished cycling, add 5 ng λ / BstEII DNA.
  
  • do not denature the λ DNA
    
• mix by pipetting up and down and incubate at 72C for 15'
  

Clean up and quantify your radiolabeled probe.

• note: Using 5 μl α-³²P-dATP (50 μCi @ 3000 Ci/mmol) and 1 μl non-radioactive dATP at 20 μM as given in this protocol is equivalent to using 6 μl of α-³²P-dATP (50 μCi @ 1370 Ci/mmol) which is a final concentration of 8.33 mCi/ml with no additional non-radioactive dATP
  

Denature the probe by boiling for 2' and snap cooling in an icewater bath for 2' immediately before using.

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References

Lawrence M. Mertz and Ayoub Rashtchian, "Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes." Analytical Biochemistry 221: 160-165 (1994)

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Comments

• α-³²P-dCTP can be used instead of α-³²P-dATP, if the other nucleotide mixes are substituted appropriately