Genotyping

The Genotyping core is available for microsatellite genotyping of the mouse and human genomes. All work is project based and designed in consultation with our technical staff.

If you have any questions, please contact our Genotyping core technician at: genotype@genome.med.harvard.edu

• Overview
• Pricing
• Submission Details

Overview

The genotyping core is available for microsatellite marker analysis using our 3730xL DNA Analyzer. The Biopolymers Facility maintains a stock of labeled primers for numerous loci covering the mouse genome and human genome at a 10cM density. Other individual markers may be purchased from ABI. Custom markers may also be used if supplied by the client or we will design order and validate primers for an additional fee. All standard primers are labeled with 6-FAM, VIC or NED dyes, and are available through Applied Biosystems.

The primers for microsatellite genotyping are designed from DNA sequences adjacent to regions of tandem repeat units. The variable length of these repeat regions produces polymorphic allele fragments during PCR that can be analyzed through capillary electrophoresis. Along with size differences we take advantage of three different fluorescent channels to run a multiplex analysis of your fragments in a single capillary.

Microsatellite Markers Available In Stock

The BPF has, in house, the entire Mouse Marker Panel and Panels 1 - 28 of the Human Linkage Mapping Set. Any primer may be ordered individually.

A complete lists of primers are available as Adobe PDF files:

• Download the Mouse Marker Set
• Download the Human Linkage Set

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Pricing

The standard service for genotyping includes a labeling PCR reaction, ROX size standard and analysis on the 3730xL. When analysis is complete your data will be posted to our secure FTP server as a ".fsa" file which can be opened with GeneMapper. For the convenience of our clients who wish to provide their own labeled products we have divided our charges in two parts: PCR and Analysis.

* Note: This is the maximum potential charge per capillary. Under normal conditions we will multiplex three labeled products in a single run using the different dye sets. Depending on size differences between the expected fragments we may run nine markers or more per capillary.

Example 1

Marker Name Size Range Dye Pool#  Marker1 91-97 NED 1  Marker2 150-154 VIC 1  Marker3 200-210 FAM 1  Marker4 98-104 FAM 1  Marker5 120-132 NED 1  Marker6 190-210 VIC 1

Since each marker in one color is more than 20bp away from the other, they can be multiplexed into one pool.

Cost: per DNA sample

Each Marker	$1.00	$6.00
Capillary Run	(1 pool)	$0.50
	Total:	$6.50
	Per marker average:	$1.08

Example 2

Marker Name Size Range Dye Pool#  Marker1 100-105 FAM 1  Marker2 102-110 FAM 2  Marker3 108-120 NED 1  Marker4 106-140 FAM 3

Since 3 of the 4 markers are FAM in overlapping sizes, they must each be put in a separate pool. The NED can multiplex with any of them.

Cost: per DNA sample

Each Marker	$1.00	$4.00
Capillary Run	(3 pools)	$1.50
	Total:	$5.50
	Per marker average:	$1.37

Primer design and validation Fees

• Primer will be deigned using primer3 software and ordered.
• When they arrive, they will be validated using a standard PCR reaction and then run on an agarose gel.
• If primers fail validation, they will be re-designed and re-ordered.
• This will continue until we have working primers, or until we do not feel any further design will be of any benefit.
• Once we have successful primers. we will order the fluorescently labeled primers.
• We will validate on a few of your DNA samples(8-16). Once we are convinced they will perform well, we will continue and complete the project.

Costs

Service Price Non-labeled primer pairs usually $10-20 Labeled forward primer usually $100-150 Primer design costs $35/hr Validation costs $0.60/reaction

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Submission Details

Templates

The final concentration of template should be 50ng/ul. For each loci you will need 1.2ul total volume. (60ng DNA)

Primers

You may select primers from our stock sets.

• Mouse
• Human

If you are not selecting from our stock sets, you have two options. You may design, order and provide the primers to us. Otherwise, primers may be designed, ordered and validated by the BPF for an additional cost.

3730xL Fragment Analysis

Clients who have fluorescently labeled microsatellite fragments using Applied Biosystems chemistry may submit whole plates for 3730xL analysis. Please re-hydrate your samples in Hi Di formamide and be sure to include a size standard. You will need to generate a plate sheet based on our template file and then e-mail your completed data table to our genotyping core at: genotype@genome.med.harvard.edu.

All 3730xL fragment analysis samples should be prepared in an ABI compatible plate such as ABgene/Marsh T0296PE with a total volume of 12ul.

Please contact us for 384 well format information.

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