Using the IMpRH DNA

INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) map

Using the IMpRH panel for Gene Mapping

Page Contents: [DNA Information] [PCR Amplification] [Agarose Gels] [Scoring and Analysis] [DNA inventory] [Gel Images] This document provides information regarding the INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) panel. In order to receive DNA from this panel the Material Transfer agreement (MTA) must be completed. Information regarding the construction of the panel and the initial IMpRH map is presented in Yerle et al. (1998) and Hawken et al. (1999) respectively. Radiation hybrid maps and information regarding these maps can also be obtained at http://fabctr.umn.edu/RHmaps/ and http://www.toulouse.inra.fr/lgc/pig/RH/IMpRH.htm.Questions regarding the RH panel can also be forwarded to IMpRH@tc.umn.edu.

Hawken, R., J. Murtaugh, G. Flickinger, M. Yerle, A. Robic, D. Milan, J. Gellin, C. Beattie, L. Schook, and L. Alexander. 1998. A first-generation radiation hybrid map of the swine genome. In Press.

Yerle, M, P. Pinton, A. Robic, A. Alfonso, Y. Palvadeau, C. Delcros, R.J. Hawken, L. Alexander, C. Beattie, L. Schook, D. Milan, J. Gellin. 1998. Construction of a whole- genome radiation hybrid panel for high-resolution gene mapping in pigs. Cytogenet. Cell Genet. 82(3-4):182-8.

DNA Information

The IMpRH panel consists of 118 samples, 5 'parental' DNA samples and hamster genomic DNA, totaling 124 tubes (refer to inventory, Table 1). You will need to provide porcine genomic DNA for positive DNA controls. DNA is supplied to you at 50µg/ml (except for the parental DNAs which have much lower concentrations). The stock concentration DNA should be kept at -20°C. DNA at user concentrations can be kept at 4°C. Excessive freeze/thawing of the DNA should be avoided. We recommend using 25ng for each PCR.

DNA from RH clones are numbered from #1 to #118 where;

IMpRH L1 to l99	were made from porcine parental DNA lymphocytes, and
IMpRH F100 to F118	were made from porcine parental DNA fibroblast.

The 'parental' DNA samples are labeled A, B, C, 4071, and 4072. These samples are DNA from pigs used to create the RHs. Correspondence between parent and IMpRH numbers is as follows:

Parent A:	IMpRH 1	to	IMpRH 13
Parent B:	IMpRH 14	to	IMpRH 22
Parent C:	IMpRH 23	to	IMpRH 99
Parent 4071:	IMpRH 100	to	IMpRH 108
Parent 4072:	IMpRH 109	to	IMpRH 118

We have provided a limited amount of DNA for these 5 parents since it is only necessary to use them if your PCR has yielded varying sized bands (alleles) for a particular marker.

We recommend dispensing 25 ng of RH DNA into each of two 96 well plates. Positive controls should be present on both plate 1 and plate 2.

PCR Amplification

Amplification conditions for individual primer pairs need to be optimized using both hamster and porcine genomic DNA. EACH MARKER MUST BE AMPLIFIED IN DUPLICATE ON THE RH PANEL IN ORDER TO ASSURE ACCURATE MAPPING.

At Minnesota, each IMpRH clone was amplified in a 15 µl reaction volume containing 25 ng of hybrid DNA, 1.5 mM MgCl2, 10 mM Tris.HCl pH9.0, 50 mM KCl, 0.1% Triton-X, 0.25U Taq and 66 µM of each dNTP. Amplification was carried out using the following conditions: denaturation at 92°C 2 min, then cycling between 92°C 30 sec, annealing temperature 30 sec, 72°C 30 sec, for 30 cycles. An extension step (5 min) at 72°C concluded the amplification reaction. Controls consisted of hamster genomic DNA, porcine genomic DNA and a negative control reaction containing no DNA.

Agarose Gels

PCR products for each marker were electrophoresed using either a 2 or 3% agarose gel in TBE buffer at 8v/cm for 20-30 minutes (based on fragment size). For each marker, two agarose gels were run (one of the duplicates per gel). Gels were photographed using an Eagle Eye II (Stratagene EagleSight version 3.0). All images were stored electronically as well as photographically. Examples of 2 agarose gels run for one marker are presented in Figure 1.

Scoring and analysis

Each marker was scored from the photos by two individuals and the results compared. Loci were scored either as present (1 or +), absent (0 or -) or ambiguous (2 or ?) for each hybrid. Ambiguous data points (hybrids which only amplified in one of the duplicates, or very faintly amplified on both) were re-amplified in duplicate. Any remaining discrepancies were scored as ambiguous. To illustrate the scoring method we used, the scoring of gels presented in Figure 1 is presented in Table 2. Initial RH maps were prepared by the University of Minnesota. The INRA toulouse web site also has created links to them. The web based program to analyze this RH data is available at http://imprh.toulouse.inra.fr/.

Table 1. Inventory of the IMpRH panel

IMpRH#	conc (µg/ml)	IMpRH#	conc (µg/ml)	IMpRH#	conc (µg/ml)	IMpRH#	conc (µg/ml)
L1	50	L39	50	L77	50	F115	50
L2	50	L40	50	L78	50	F116	50
L3	50	L41	50	L79	50	F117	50
L4	50	L42	50	L80	50	F118	50
L5	50	L43	50	L81	50	Wg3	50
L6	50	L44	50	L82	50	A	38
L7	50	L45	50	L83	50	B	37
L8	50	L46	50	L84	50	C	43
L9	50	L47	50	L85	50	4071	13
L10	50	L48	50	L86	50	4072	43
L11	50	L49	50	L87	50
L12	50	L50	50	L88	50
L13	50	L51	50	L89	50
L14	50	L52	50	L90	50
L15	50	L53	50	L91	50
L16	50	L54	50	L92	50
L17	50	L55	50	L93	50
L18	50	L56	50	L94	50
L19	50	L57	50	L95	50
L20	50	L58	50	L96	50
L21	50	L59	50	L97	50
L22	50	L60	50	L98	50
L23	50	L61	50	L99	50
L24	50	L62	50	F100	50
L25	50	L63	50	F101	50
L26	50	L64	50	F102	50
L27	50	L65	50	F103	50
L28	50	L66	50	F104	50
L29	50	L67	50	F105	50
L30	50	L68	50	F106	50
L31	50	L69	50	F107	50
L32	50	L70	50	F108	50
L33	50	L71	50	F109	50
L34	50	L72	50	F110	50
L35	50	L73	50	F111	50
L36	50	L74	50	F112	50
L37	50	L75	50	F113	50
L38	50	L76	50	F114	50

Figure 1. Photograph of two agarose gels for the amplification of the IMpRH panel using primers for the alpha-microglobulin-bukunin precursor protein (AMBP). Scoring for these gels is presented above each lane and summarized in Table 2.

Amplification of AMBP No.1

Amplication of AMBP No.2

Table 2. Scoring for AMBP

Gel lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 +ve

Gel#1 0 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1

Gel#2 0 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1

summary 0 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

Gel lane 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 +ve

Gel#1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

Gel#2 0 0 1 0 0 0 0 0 0 0 0 0 0 0 vvf 0 0 0 0 0 0 0 0 0 1

summary 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0

Gel lane 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 +ve

Gel#1 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 f 1 1 0 0 0 0 0 1

Gel#2 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 0 0 0 0 1

summary 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 0 0 0 0

Gel lane 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 +ve

Gel#1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 vvf 0 0 1 0 0 1

Gel#2 0 0 0 0 0 1 0 f 0 0 1 0 0 0 0 0 0 1 f 0 0 1 0 0 1

summary 0 0 0 0 0 2 0 2 0 0 1 0 0 0 0 0 0 1 2 0 0 1 0 0

Gel lane 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 R +ve -ve

Gel#1 0 0 0 1 0 0 1 0 f 0 0 0 0 0 0 0 0 1 1 0 1 1 0 1 0

Gel#2 0 0 0 1 0 0 1 0 f 0 0 0 0 0 0 0 0 1 1 0 1 1 0 1 0

summary 0 0 0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 1 1 0 1 1

Gel lane: +ve- pig DNA control; -ve-no DNA control; R-Rodent DNA control;
Scoring: 1-positive; 0-negative; f-faint amplification; vf-very faint amplification; vvf-very very faint amplification; 2-ambigous data point (needs to be repeated).
For this marker, RH sample # 39, 78, 80 and 91 should be repeated.

Gel lane	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	+ve
Gel#1	0	0	1	0	1	0	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	1
Gel#2	0	0	1	0	1	0	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	1
summary	0	0	1	0	1	0	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0

Gel lane	25	26	27	28	29	30	31	32	33	34	35	36	37	38	39	40	41	42	43	44	45	46	47	48	+ve
Gel#1	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
Gel#2	0	0	1	0	0	0	0	0	0	0	0	0	0	0	vvf	0	0	0	0	0	0	0	0	0	1
summary	0	0	1	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0	0	0

Gel lane	49	50	51	52	53	54	55	56	57	58	59	60	61	62	63	64	65	66	67	68	69	70	71	72	+ve
Gel#1	0	0	0	0	1	0	1	0	0	0	1	0	0	0	0	0	f	1	1	0	0	0	0	0	1
Gel#2	0	0	0	0	1	0	1	0	0	0	1	0	0	0	0	0	1	1	1	0	0	0	0	0	1
summary	0	0	0	0	1	0	1	0	0	0	1	0	0	0	0	0	1	1	1	0	0	0	0	0

Gel lane	73	74	75	76	77	78	79	80	81	82	83	84	85	86	87	88	89	90	91	92	93	94	95	96	+ve
Gel#1	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	1	vvf	0	0	1	0	0	1
Gel#2	0	0	0	0	0	1	0	f	0	0	1	0	0	0	0	0	0	1	f	0	0	1	0	0	1
summary	0	0	0	0	0	2	0	2	0	0	1	0	0	0	0	0	0	1	2	0	0	1	0	0

Gel lane	97	98	99	100	101	102	103	104	105	106	107	108	109	110	111	112	113	114	115	116	117	118	R	+ve	-ve
Gel#1	0	0	0	1	0	0	1	0	f	0	0	0	0	0	0	0	0	1	1	0	1	1	0	1	0
Gel#2	0	0	0	1	0	0	1	0	f	0	0	0	0	0	0	0	0	1	1	0	1	1	0	1	0
summary	0	0	0	1	0	0	1	0	1	0	0	0	0	0	0	0	0	1	1	0	1	1