the technical convenience of milk as a source of dna can be expected to increase the field of application of the markerbased methods for genetic analysis of goat genome . it has a remarkable importance for several reasons related to governmental regulation and public health . mixture in dairy products and species substitution should be observed to be a cause of human adverse reactions ( bottero et al , 2003 ) . the source of dna in milk could be from somatic cells ( fahr et al , 1999 ) . goat milk is similar to cow milk with around 87 percent water , 67 percent energy , 3.3 percent protein , 4.0 percent fat , and 4.6 percent carbohydrates ( belanger and jerry , 2001 ) . goat milk differs from cow and human milk in several ways , among them higher digestibility and lower lactose ( larson , 1978 ) . one difference is that goat milk has smaller fat globules present due to the lack of the enzyme that aggregates the globules in the milk ( belanger and jerry , 2001 ) . the objective of the present study was to evaluate milk as a source of goat dna and as a substrate for pcr amplification using mitochondrial cytochrome - b gene as a target dna for pcr amplification . this was prospective study in which samples are collected throughout the study this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010 25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 , 12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml . the primers were used at a concentration of 20mole / l which appears to 2 l . next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing . this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units . the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes . thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) . the 10x tris borate edta ( tbe ) buffer was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml . 15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed . this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010 25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included . before collection , the teats were cleaned with alcohol to avoid samples contamination from skin . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . before collection , the teats were cleaned with alcohol to avoid samples contamination from skin . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 , 12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml . the primers were used at a concentration of 20mole / l which appears to 2 l . next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing . this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units . the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes . thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) . all pcr amplification product samples were visualized using agarose gel electrophoresis . the 10x tris borate edta ( tbe ) buffer was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml . 15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed . the dna yield varies with the different stages of lactation as compared with the yield from peripheral blood leukocytes . the pcr - based assay described in this study afforded sensitive and specific identification of goat mtcyt - b dna . using the pair of primers ( gsl1 , gsr2 ) . the sensitivity studies indicate that , the 428bp pcr products were detected from not < 1.0 pg of goat mtcyt - b gene ( fig.1 ) . the specificity studies for goat primers indicate that the described pcr assay failed to amplify the specific pcr product from dna extracted from other animal species including cattle , sheep , swine , camel , deer , horse , donkey , and human ( figure 2 ) . using primers gsl1 , gsr2 , amplification of the goat - specific 428 bp pcr product was produced from different milk samples collected from goats included in this study ( figure 3 , 4 , 5 ) . in goat , however , obvious technical difficulties exist in the collection of blood samples from large numbers of individuals among widely separated herds , limiting the application of marker - based methods for genetic analysis and for genetic improvement of economic traits in dairy goat . the scientific data presented in this study indicate that dna extracted from goat milk could serve as substrates for pcr amplification of the full length of the caprine mitochondrial cytochrome - b gene or a fragment of the gene . since collection of milk is a non invasive procedure , it can often substitute for blood as a source of dna . the technical convenience of milk as a source of dna can be expected to increase the field of application of the marker - based methods for genetic analysis of goat genome . it has a remarkable importance for several reasons related to governmental regulation and public health . protection against species substitution or admixture in dairy products is of significant importance ( bottero et al , 2003 ) . it was found that most milk proteins , even at low concentration , are potential allergens ( sampson , 2003 ) . also , cow s milk was reported as the main dairy product responsible for human adverse reaction ( rance et al , 2005 ) . thus , the counterfeiting of goat s milk with cow s milk may be considered as a health risk making species identification an important issue in current food safety requirement . the common fraudulent practice found in the dairy production line is the use of a cheaper type of milk in substitution of more expensive ones . currently , different methods are used for species identification in milk and milk products including immunological ( addeo et al , 1995 ) , electrophoretic ( cartoni et al , 1998 ) , and chromatographic techniques ( pellegrino et al , 1991 ) . among these methods , capillary electrophoresis , two dimensional electrophoresis , isoelectric focusing of milk caseins which is the european community reference method for cow s milk detection ( ecr , 1996 ) , however these methods ca nt always distinguish milk from closely related species and not suitable for heat treated milk . the pcr amplification technology , described in this study , provides a simple , rapid , reliable and sensitive method for species identification and differentiation . the time required for the pcr amplification was approximately 3 hours ; this means that confirmatory diagnosis could be obtained within the same working day . ( 1993 ) reported that milk is less reliable source of dna than is blood because it requires large size of sample and high concentration of somatic cells ( e. lipkin et al , 1993 ) . but in our study we used only 200l of milk sample for extraction of dna , and the pcr products were the same in length and approximate quantity for milk , for dna extracted from milk , and for dna extracted from blood , this difference may reflect the detection procedures described above . the intensity of dna signal in gel electrophoresis , show that , there is a decline in the dna content in the late lactation periods . this may indicate the milk dna content is high in the first and second lactation periods and then declines . for more significant results dna quantification by spectrophotometer these non - nuclear targets possess several advantages over nuclear genes ( unseld et al,1995 ) . they are generally more abundant in any given sample than single - copy nuclear genes , and , because mitochondrial dna has a relatively high mutation rate compared with the nuclear dna , they contain a greater accumulation of point mutations that can be used to better define species differences . moreover , mitochondrial dna tends to be inherited through the maternal germ line , and the resulting lack of heterozygosity in the alleles under study simplifies analysis ( kocher et al , 1989 ) . the mtcyt - b dna was selected in this work as the target sequence for species identification . pcr - based assay described in this study would be advantageous in the variety of conditions including comparative genomics , species identification in milk and milk products , experimental physiology and can be recommended in the quality control departments in order to support policies and regulation of import / export of milk and milk products .