18 Quantitative DNA Fiber Mapping
241
Procedure
1. Cloning vector-specific probes
The generation of Pl/PAC-, BAC- and YAC-vector probe DNA takes advantage of the access to published vector sequences. PCR primers are typically designed to amplify fragments of 1100-1400 bp of vector sequence.
Several such oligonucleotide pairs have been designed in several laboratories including ours and are used in either single pairs or combinations.
The PCR usually follows standard conditions, i.e., a Tris-HCI buffer containing 1.5 mM MgClz and 1 unit Taq DNA polymerase per 50 III reaction is
used, annealing temperatures range from 50°C to 60°C.
On the other hand, the YAC cloning vectors pJs97 and pJs98, cloned in
plasmid vectors (BRL), can be used to prepare probes useful to determine
the orientation of the YAC insert (Duell et aI., 1997). For this purpose,
plasmid DNA is extracted using the above alkaline lysis protocol or a commercial kit and labeled by random priming as described below.
2. Mixed base oligonucleotide primed PCR
The DNA probes for counterstaining the YAC DNA fibers are generated by
mixed base oligonucleotide primed PCR (sometimes referred to as degenerate oligonucleotide primed PCR or 'DOP-PCR') (Cassel et aI., 1997). An
aliquot of the HMW DNA obtained by PFGE for fiber preparation is PCR
amplified for a total of 42 cycles with oligonucleotide primers that anneal
about every 200-800 nucleotides. In our preferred scheme, we use two different DNA amplification programs. Initially we perform a few manual
PCR cycles using T7 DNA polymerase to extend the oligonucleotide primers at a relatively low temperature. Next, DNA copies prepared in these
first cycles are amplified using the thermostable Taq DNA polymerase and
a rapid thermal cycling scheme.
In the first amplification stage, T7 DNA polymerase ('Sequenase II',
Amersham Pharmacia Biotech) is used in 5-7 cycles to extend the mixed base primer JUNI (5-CCAAGCTTGCATGCGAATTCNNNNCAGG-3,
N=ACGT) that is annealed at low temperature. Briefly, 2-3 III of HMW
DNA solution are removed from the bottom of each tube and PCR amplified using the following conditions: denaturation at noc for 3 min, primer annealing at 20°C for 2 min and extension at 37°C for 6 min. Sequenase must be added after each denaturation. (See Chapter 29 by Fung et al.
for details.)
241
Procedure
1. Cloning vector-specific probes
The generation of Pl/PAC-, BAC- and YAC-vector probe DNA takes advantage of the access to published vector sequences. PCR primers are typically designed to amplify fragments of 1100-1400 bp of vector sequence.
Several such oligonucleotide pairs have been designed in several laboratories including ours and are used in either single pairs or combinations.
The PCR usually follows standard conditions, i.e., a Tris-HCI buffer containing 1.5 mM MgClz and 1 unit Taq DNA polymerase per 50 III reaction is
used, annealing temperatures range from 50°C to 60°C.
On the other hand, the YAC cloning vectors pJs97 and pJs98, cloned in
plasmid vectors (BRL), can be used to prepare probes useful to determine
the orientation of the YAC insert (Duell et aI., 1997). For this purpose,
plasmid DNA is extracted using the above alkaline lysis protocol or a commercial kit and labeled by random priming as described below.
2. Mixed base oligonucleotide primed PCR
The DNA probes for counterstaining the YAC DNA fibers are generated by
mixed base oligonucleotide primed PCR (sometimes referred to as degenerate oligonucleotide primed PCR or 'DOP-PCR') (Cassel et aI., 1997). An
aliquot of the HMW DNA obtained by PFGE for fiber preparation is PCR
amplified for a total of 42 cycles with oligonucleotide primers that anneal
about every 200-800 nucleotides. In our preferred scheme, we use two different DNA amplification programs. Initially we perform a few manual
PCR cycles using T7 DNA polymerase to extend the oligonucleotide primers at a relatively low temperature. Next, DNA copies prepared in these
first cycles are amplified using the thermostable Taq DNA polymerase and
a rapid thermal cycling scheme.
In the first amplification stage, T7 DNA polymerase ('Sequenase II',
Amersham Pharmacia Biotech) is used in 5-7 cycles to extend the mixed base primer JUNI (5-CCAAGCTTGCATGCGAATTCNNNNCAGG-3,
N=ACGT) that is annealed at low temperature. Briefly, 2-3 III of HMW
DNA solution are removed from the bottom of each tube and PCR amplified using the following conditions: denaturation at noc for 3 min, primer annealing at 20°C for 2 min and extension at 37°C for 6 min. Sequenase must be added after each denaturation. (See Chapter 29 by Fung et al.
for details.)