DRB3 PCR-RFLP analysis
The availability of DNA sequence information for class II genes
enabled the development of PCR-based typing methods.
In cattle, restriction site polymorphism is correlated with the
high degree of sequence polymorphism in DRB3 exon 2, thus enabling
the combined use of PCR and RFLP for detection of DRB3 alleles.
The strategy developed for PCR-RFLP of DRB3 alleles was to select
the minimum number of enzymes that discriminate the maximum number
of alleles. To date, 50 different types have been identified in
a 284-bp segment of DRB3 exon 2 using RsaI, BstY1 and HaeIII but
not all of the types have a corresponding sequenced allele. Two
restriction sites are found in codons for peptide binding residues.
In the 5th BoLA workshop, PCR-RFLP was shown to be the most powerful
DRB3 typing method and was extremely useful for haplotype discrimination
(see the table of class II haplotypes in the 5th workshop report).
Although the method works best when family data are available,
population studies are feasible because of the minimal overlap
in fragment patterns. Not unexpectedly, not all DRB3 alleles can
be discerned by PCR-RFLP; however, the method can easily be extended
by adding more enzymes for alleles not split by RsaI, BstY1 and
HaeIII.
This text was taken from Lewin
1996, with permission from the publisher
CRC Press.
Restriction
patterns RsaI
Patterns
are from:
78 33 30 39 54 50
a ____________________|_______|_______|__________|_____________|__________
111 30 39 54 50
b ____________________________|_______|__________|_____________|__________
111 30 93 50
c ____________________________|_______|________________________|__________
111 30 143
d ____________________________|_______|___________________________________
141 39 51 50
e ____________________________________|__________|__\/_________|__________
141 39 54 50
f ____________________________________|__________|_____________|__________
141 39 104
g ____________________________________|__________|________________________
111 69 54 50
h ____________________________|__________________|_____________|__________
180 54 50
i _______________________________________________|_____________|__________
78 63 93 50
j ____________________|_______________|________________________|__________
78 156 50
k ____________________|________________________________________|__________
234 50
l _____________________________________________________________|__________
111 69 104
m ____________________________|__________________|________________________
180 104
n _______________________________________________|________________________
284
o ________________________________________________________________________
111 30 39 51 50
p ____________________________|_______|__________|_\/__________|__________
141 90 50
q ____________________________________|____________\/__________|__________
111 30 90 50
r ____________________________|_______|____________\/__________|__________
141 93 50
s ____________________________________|________________________|__________
141 143
t ____________________________________|___________________________________
111 123 50
u ____________________________|________________________________|__________
78 102 54 50
v ____________________|__________________________|_____________|__________
78 33 69 54 50
w ____________________|_______|__________________|_____________|__________
78 33 69 104
x ____________________|_______|__________________|________________________
78 63 39 54 50
y ____________________|_________________|________|_____________|__________
BstYI
Patterns
a - e are from van Eijk
et al. 1992.
199 85
a ____________________________________________________|___________________
284
b ________________________________________________________________________
196 85
c _________________________________________________\/_|___________________
87 197
d _____________________|__________________________________________________
87 112 85
e _____________________|______________________________|___________________
HaeIII
Patterns
are from
- a
- f - van Eijk et al.
1992
- g
+ h - Gelhaus et al. 1995
- i
- based on allele DRB3*25011.
167 52 65
a _________________________________________|________________|_____________
219 65
b __________________________________________________________|_____________
167 49 65
c _________________________________________|_______\/_______|_____________
190 29 65
d ____________________________________________________|_____|_____________
167 117
e _________________________________________|______________________________
167 4 48 65
f _________________________________________|_|______________|_____________
164 55 65
g _______________________________________|__________________|_____________
167 46 6 65
h ___________________________________________|___________|__|_____________
167 4 113
i _________________________________________|_|____________________________
PCR
methods
Amplification
of BoLA-DRB3
Two stage amplification.
First stage primers:
- HL030
5'-ATCCTCTCTCTGCAGCACATTTCC-3'
Contains 7 nucleotides of the 5' and 3' ends of exon 2 plus
intron sequences.
- HL031
5'-TTTAAATTCGCGCTCACCTCGCCGCT-3'
Contains 8 nucleotides of the 5' and 3' ends of exon 2 plus
intron sequences.
Second
stage primers:
- HL030
5'-ATCCTCTCTCTGCAGCACATTTCC-3'
- HL032
5'-TCGCCGCTGCACAGTGAAACTCTC-3'
Consists entirely of nucleotides of the 3' end of exon 2
and has an 8 basepair overlap with 3' end of HL031.
First
stage cycle profile:
- Denaturation:
4 minutes at 94C
- 10
cycles of 1 minute at 94C, 2 minutes at 60C and 1 minute
at 72C
- Final
extension: 5 minutes at 72C
Second
stage cycle profile:
- 25
cycles of 1 minute at 94C, 30 seconds at 65C
- Final
extension: 5 minutes at 72C
Restriction
Analysis
Detection
of BoLA-DRB3 alleles
10ul of each PCR product is digested with 5 units of RsaI
(at 37C), HaeIII (at 37C) and BstYI (at 50C) for 1-2 hours.
Restriction fragments resolved by 6% polyacrylamide gel electrophoresis,
with size standards of MspI or HaeIII digested pBR322.
Fragments were visualised after staining with ethidium bromide.
PCR-RFLP
patterns
Please note that while the vast majority of the PCR-RFLP types
listed here are based on the predicted patterns found in alleles
defined in BoLA workshops, a few correspond to alleles which
have not been sequenced. Some of these patterns were defined
in population studies, and others were predicted from sequences
which are not yet confimed as DRB3 alleles.
Types
1 - 40 are as described in van
Eijk et al. 1992 and Gelhaus
et al. 1995, while 41 - 54 are described in Maillard
et al. 1999.
