Methods in Molecular Biology • 16 Enzymes of Molecular Biology

48 Landgraf and Wolfes

Single strand breaks are generated In a footprintlng experiment or

using Maxam Gilbert chemistry

I

Annealing of gene specific pdmers and

extenUon with DNA polymerase |

V

I

T4 Iigase mediated blunt end Ilgatlon

of ollgodeoxynucleotide linkers [

f~ ( I'l t

I(((I

PCR amplification I

I{{i

h" ( ¢l

itcd

i{(11

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l.. .. ,a

Sequencing gel

Fig. 6. Application of PCR to in vivo footprinting and detection of methylation pattern.

Although the patients were cytogenetically inconspicious even 3-6 yr
after surgery, CML-specific transcripts could be detected by PCR of the
reverse transcribed RNA (74).
If the boundaries of genes are not exactly known, ligation of specific
sequences to the cDNA will render an amplification feasible. Primers
complementary to the ligated oligodeoxynucleotides permit the PCR