Page 6 - IJMZ-Vol.3-No.3
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Intl. J. of Molecular Zoology, 2013, Vol.3, No.3, 10
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13
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be concluded that the animals of BB genotype for
higher lactation length and AB genotype for lactation
yield and animals of AA genotype for less service
period may be used for future breeding.
3
Materials and Methods
The present study comprised of 54 blood samples of
Frieswal lactating cows collected from military dairy
farm, Jabalpur, along with their lactation length,
lactation yield and service period records. Genomic
DNA was extracted from venous blood by the method
of John et al (1991) with slight modification. Purity
and concentration of genomic DNA was determined
by using Nano-drop spectrophotometer. Genomic
DNA quality was assessed by using 0.8% horizontal
submarine agarose electrophoresis. PCR products
were amplified using Oligonucleotide primers specific
to bovine
PRL
gene locus were custom synthesized by
Ocimum Biosciences, Bangalore in accordance with
Lewin et al (1992) primer sequences: forward
(5′-
CGAGTCCTTATGAGCTTGATTCTT-3′)
and
reverse (5′-GCCTTCCAGAAGTCGTTTGTTTTC-3′)
primers.
Polymerase chain reaction was carried out in a final
reaction volume of 25µL. PCR reaction mixture used
for amplification of DNA was 2X PCR master mix
12.5
µL, forward primer (10 pmol/µL) 1.0 µL, reverse
primer (10 pmol/µL) 1.0 µL, genomic DNA 3.0 µL
and DNAase free water 7.5 µL. Amplification was
performed in PCR thermal cycler programmed for 32
cycles with an initial denaturation at 95
℃
for 10 min,
denaturation at 95
℃
for 1 min, annealing at 56
℃
for
1
min and extension at 72
℃
for 1 min with a final
extension at 72
℃
for 10 min. Total 5 µL of amplified
PCR product of each sample was mixed with 1 µL of
6
X gel loading dye buffer from each tube. The samples
were loaded on 2% agarose gel containing ethidium
bromide (1% solution @ 5µL/100mL) along with
80
bp DNA ladder at a constant voltage of 80 V for 30
min. in 0.5X TBE buffer. Amplified DNA was
digested with the
Rsa
I enzyme. Restriction digestion
of the PCR product was performed in a total 30 µL.
Content having 10X Buffer Tango 2 µL, PCR reaction
mixture 10 µL, restriction enzyme (10units/µL) 1 µL
and 17 µL nuclease free water. The reaction mixture
was spined for few seconds for uniform mixing and
then incubated at 37
℃
for 4 h and 30 min in the
water bath. After restriction digestion, the restriction
product mixtures were electrophoresed on 3.5%
agarose gel containing 1% ethidium bromide @ 5
µL/100mL at constant voltage of 80 V for 80 min
using 0.5X TBE buffer. Gel loading dye (6X) was
used to load the digested PCR samples. DNA ladder
(
Range 100-1000 bp) was used as a molecular size
marker. The DNA PCR-RFLP bands were visualized
under UV light and documented by gel documentation
system. The band size was judged by comparing with
molecular size marker and recorded. Genotyping of
PRL
gene locus was carried out according to the band
pattern of respective genotypes.
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