This is a description of the protocol we use to assay for the presence of an STS for radiation hybrid mapping.

We recently changed this protocol to accomodate the adoption of AmpliTaq Gold in our PCR reactions. This new enzyme has increased our success rate by reducing the number of STSs that fail due to background. The protocol below includes changes in the PCR mixture and thermocycling conditions to allow for the use of Amplitaq Gold.

A source list of tools and materials is also available.

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Contents

• PCR Mixture
• Thermocycling conditions
• Gel Information
• Electrophoresis
• Gel Imaging
• Analysis
• Email service for localization

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PCR Mixture (per reaction)

5.0 uL	radiation hybrid DNA (at 5 ng/ul)
2.0 uL	5x buffer
0.8 uL	2.5 mM dNTP's
0.8 uL	10 uM oligonucleotide primer pair
0.07 uL	AmpliTaq Gold (5 U/uL)
1.33 ul	dH20

5X Buffer:

50 mM	Tris-HCL, pH 8.3
250 mM	KCl
12.5 mM	MgCl2

Ambiguities in typing will decrease the likelihood of obtaining a lod 6 or greater link between loci. We strongly suggest the use of duplicate typings. If after duplicate typings there are more than 7 ambiguities between the two typings, the STS shoud be reassayed under different conditions or redesigned.

Thermocycling Conditions

PCR is carried out in Perkin-Elmer 9700 Thermocyclers.

 Temp     Time (min:sec)
 95C      10:00 
 94C      0:30       | 60C      0:30       |30x  72C      0:23       | 
 72C      3:30 
 4C      Forever 

After amplification, add 5 uL of 3x loading dye to each reaction.

3X Loading Dye:

10% (w/v)	Ficoll 400
0.1 M	EDTA pH 8.0
0.025% (w/v)	Bromophenol blue

Gel Information

100 mL of
3% SeaPlaque Agarose
1x TBE

is poured in a 10 x 15 cm tray with four 26 well combs. 10 uL of each reaction is loaded into the wells using a 12 channel pipette. Msp1 digested pBR322 marker is loaded into the outside wells of each tier.

Electrophoresis

Gels are electrophoresed in 1X TBE at 120 Volts for 45 minutes.

Stain
Stain in 0.9 ug/L EtBr, 1x TBE for 15 minutes
De-Stain in 1x TBE for 30 minutes

Gel Imaging

Images of gels are acquired using a UV lightbox and 640 x 480 pixel CCD camera. TIFF formatted images are saved directly to a UNIX workstation and thermal paper hard copies are stored in notebooks.

Analysis

Analysis takes place on UNIX workstations running a modified version of K. Clark's DNA/GUI. Gels are scored in a semi-automated manner for the presence or absence of positive PCR products at the expected size. An STS must be found to be present on each of the duplicate gels to be scored as positive. Discrepancies between the duplicate gels are discarded before subsequent analysis. Assays which result in more than seven discrepancies are repeated under different conditions or primers are redesigned.

Map Construction
Once a significant number of STS's have been assayed against the hybrids for a chromosome or region of interest, semi-automated map construction can be accomplished. By using a Simulated Annealing based algorithm,   developed by Kate McKusick at SHGC, each STS is placed into LOD 6 two-point linkage bins and 1000:1 odds order bins.

Email service for random marker localization

1878 markers from the Généthon linkage map were chosen based on their distribution across the genome and assayed against the publicly available RH DNA's. See the RH Server which provides an e-mail service for localization of markers to this set.

View an abstract associated with the publicly available RH DNA's.

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