PCR amplification of ovine somatotropin gene and PCR amplification of ovine somatotropin gene and its partial characterizationits partial characterization

Moazzam Ali & Dr. Waheed AkhtarMoazzam Ali & Dr. Waheed Akhtar

SomatotropinSomatotropin

Somatotropin or growth hormone is a peptide hormone, it Somatotropin or growth hormone is a peptide hormone, it causes growth of almost all tissues of body, increases causes growth of almost all tissues of body, increases protein synthesis, mobilization of fatty acids and decreases protein synthesis, mobilization of fatty acids and decreases glucose utilization GH has remarkable biotechnological glucose utilization GH has remarkable biotechnological applications. applications.

Intron Exon

3rd PCR product

2nd PCR product

1st PCR product

Relative positions of PCR products amplified by first, second and third primer pairs. The complete gene shown at the top is 1631bp, first PCR product is 483bp, second is 414bp and third is 567 bp.

Primer designingPrimer designing

Collect sequences of growth hormone gene of sheep, goat and Collect sequences of growth hormone gene of sheep, goat and bovine animals (complete gene, partial gene, mRNA)bovine animals (complete gene, partial gene, mRNA)

All the sequences were aligned using Clustal X software All the sequences were aligned using Clustal X software

The alignments were studied for conserved regionsThe alignments were studied for conserved regions

Primers were designed in those regions using primer 0.5 and Primers were designed in those regions using primer 0.5 and primer 3 softwares. primer 3 softwares.

Finally the primers were tested using BLAST for there Finally the primers were tested using BLAST for there performance.performance.

Sequences of designed primers along with there profile

Primers Sequence Tm GC% Length

First left First right

CTGCTCCTGGCTTTCACC GCTGGGCCTCATTCTTGC

59.50 61.89

61.1% 61.1%

18bp 18bp

Second left Second right

CCAGAACACCCAGGTTGC CCGAGGTGCCAAACACC

60.09 61.12

61.1% 64.7%

18bp 17bp

Third left Third right

ATCCAGTCGTGGCTTGG TTCATGACCCTCAGGTACG

58.03 56.99

58.82 52.63

17bp 19bp

PCR amplificationPCR amplification

There is no universal protocol for PCR amplification There is no universal protocol for PCR amplification and one has to optimize reagent and temperature and one has to optimize reagent and temperature cycling steps for each set of primerscycling steps for each set of primers

PCR Results of First Primer PairPCR Results of First Primer Pair

Lanes 2 to 7 contain PCR Lanes 2 to 7 contain PCR products amplified with products amplified with increasing magnesium conc increasing magnesium conc (2 – 4.5 mM) and lane 10 is (2 – 4.5 mM) and lane 10 is size markersize marker

PCR product size is 483bp PCR product size is 483bp

1 2 3 4 5 6 7 8 9 10 11 12

PCR Results Using Second Primer PairPCR Results Using Second Primer Pair

Lanes 3 to 9 contain PCR Lanes 3 to 9 contain PCR products amplified by products amplified by increasing Mg++ conc (2 - increasing Mg++ conc (2 - 5.0 mM) and lane 11 is size 5.0 mM) and lane 11 is size markermarker

mispriming bands below the mispriming bands below the actual bands in lanes 7, 8 actual bands in lanes 7, 8 and 9and 9

PCR product size is 414bp PCR product size is 414bp   

  

1 2 3 4 5 6 7 8 9 10 11 12

Use of PCR enhancers for amplification by second Use of PCR enhancers for amplification by second primer pairprimer pair

BSA concentrationsBSA concentrations

10, 25 and 5010, 25 and 50µl/mlµl/ml

Triton X100 concentrationsTriton X100 concentrations

0.05% and 0.1%0.05% and 0.1%

BSA Triton X100

Use of PCR enhancers for amplification by second Use of PCR enhancers for amplification by second primer pairprimer pair

Betaine Conc.Betaine Conc.

1.2M1.2M

DMSO Conc.DMSO Conc.

5%5%

Glycerol Conc.Glycerol Conc.

5%5%

Betaine DMSO Glycerol

Complete ovine somatotropin gene 1.32kb PCR fragment 2nd PCR product

Nested PCRNested PCR

Amplification of 1.32kb fragment (external round of Amplification of 1.32kb fragment (external round of nested PCR)nested PCR)

Gel image showing 1321 bp Gel image showing 1321 bp PCR products amplified PCR products amplified from two different sheep from two different sheep samplessamples

Amplification of 414 bp fragment (internal round of nested PCR)Amplification of 414 bp fragment (internal round of nested PCR)

Gel image showing 414 bp PCR Gel image showing 414 bp PCR products amplified from using products amplified from using second primer pair and 1.32kb second primer pair and 1.32kb PCR product as template from PCR product as template from three different sheep samplesthree different sheep samples

PCR Results Using Third Primer PairPCR Results Using Third Primer Pair

Lanes 2-7 contained PCR Lanes 2-7 contained PCR products amplified under products amplified under increasing magnesium conc (2 - increasing magnesium conc (2 - 4.5 mM with 0.5 mM increments) 4.5 mM with 0.5 mM increments) and lanes 8-9 under increasing and lanes 8-9 under increasing betaine (0.5 and 1 M)betaine (0.5 and 1 M)

Nested PCR improved Nested PCR improved amplification, results not shown amplification, results not shown herehere

PCR product size is 567 bpPCR product size is 567 bp

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction digestion of amplified productsRestriction digestion of amplified products

Theoretical patterns of restriction digests were Theoretical patterns of restriction digests were determined using appropriate softwares and then the determined using appropriate softwares and then the patterns were confirmed by restriction digestionpatterns were confirmed by restriction digestion

Some complications in restrictionSome complications in restriction

Gene duplication at growth hormone locus and it is an Gene duplication at growth hormone locus and it is an allelic phenomenonallelic phenomenon

Partial digestion and star activityPartial digestion and star activity

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by First Primer PairFirst Primer Pair

Uncut, lane 2Uncut, lane 2

Mva Mva I,I, lanes 3, 4, 5lanes 3, 4, 5

Pvu Pvu II,II, lanes 6, 7, 8lanes 6, 7, 8

Bsu Bsu RI,RI, lanes 9, 10, 11lanes 9, 10, 11

Size marker, lanes 1, 12Size marker, lanes 1, 12 Three different samples wereThree different samples were

studiedstudied

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by Second Primer PairSecond Primer Pair

Uncut, lane 2Uncut, lane 2

Apa Apa I, lanes 4,5,6I, lanes 4,5,6

Bsu Bsu RI, lanes 7,8,9RI, lanes 7,8,9

Size marker, lane 11Size marker, lane 11

Three different samplesThree different samples

were studiedwere studied

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by Second Primer PairSecond Primer Pair

Uncut, lane 2Uncut, lane 2

MvaMva I, lanes 4,5,6 I, lanes 4,5,6

PstPst I, lanes 7,8,9 I, lanes 7,8,9

Size marker, lane 11Size marker, lane 11

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by Third Primer PairThird Primer Pair

Uncut, lane 2Uncut, lane 2

SmaSma I, lanes 4,5,6 I, lanes 4,5,6

MvaMva I, lanes 7,8,9 I, lanes 7,8,9

Size marker, lane 11Size marker, lane 11

Three different samplesThree different samples

were studiedwere studied

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by Third Primer PairThird Primer Pair

Uncut, lane 2Uncut, lane 2

ApaApa I, lanes 4,5,6 I, lanes 4,5,6

BspBsp 1431, lanes 7,8,9 1431, lanes 7,8,9

Size marker, lane 11Size marker, lane 11

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Digestion of PCR Product, Amplified by Restriction Digestion of PCR Product, Amplified by Third Primer PairThird Primer Pair

Uncut, lane 2Uncut, lane 2

BsuBsu RI, lanes 4,5,6 RI, lanes 4,5,6

PstPst I, lanes 7,8,9 I, lanes 7,8,9

Size marker, lane 11Size marker, lane 11

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

Restriction Map of Ovine Growth Hormone GeneRestriction Map of Ovine Growth Hormone Gene

Puv II (star activity site) Bsu RI Pst I Bsp 1431 Bsu RI (Polymorphic) Puv II (star activity site) Mva I Mva I Bsu RI Mva I Apa I Bsu RI Apa I Mva I Bsu RI Bsu RI Bsu RI Mva I Bsu RI Mva I Bsu RI Pvu II Mva I Bsu RI Bsu RI Bsp 1431

Thank youThank you