Nucleic acid Blotting Technique

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Blotting Technique:

1 Presented by- Salunkhe Pankaj S. M.Pharm 2 nd sem. Department Of Pharmacology R.C.Patel College of Pharmacy,Shirpur Blotting Technique SHRI

Content:-:

2 Content:- Basic related to DNA DNA storage and collection History and Background regarding blotting Basic Technique used in Blotting -Isolation and Purification -Gel Electrophoresis -Hybridization -Washing -Detection Application Reference

DNA1,5,7:

3 DNA 1,5,7 Each individuals unique genetic blueprint is stored in material known as DNA. DNA is found in all cells containing a nucleus. DNA can be extracted for analysis from hair, bones, saliva, sperm, skin, organs, all body tissues and blood.

DNA1,5,7:

4 DNA 1,5,7 The deoxyribonucleic acid, DNA, is a long chain of nucleotides which consist of: 1. Deoxyribose(sugar with 5 carbons) 2. Phosphate groups 3. Organic(nitrogenous)bases

Nitrogenous Bases1,5,7,14:

5 Nitrogenous Bases 1,5,7,14 Two classes: Purines Adenine Guanine Pyrimidines Cytosine Thymine

DNA1,5,7:

6 DNA 1,5,7 The base adenine (A) always pairs with thymine (T). The base guanine (G) always pairs with cytosine (C).

DNA1,5,7:

7 DNA 1,5,7 Example First strand GGGTTTAAACCC Second strand CCCAAATTTGGG 6

DNA STORAGE AND COLLECTION19,20:

8 DNA STORAGE AND COLLECTION 19,20 I. Temperature Storage for DNA Purified DNA may be refrigerated at 4°C for up to 3 years. Samples kept over 3 years should be frozen at -70°C.

DNA STORAGE AND COLLECTION19,20:

9 DNA STORAGE AND COLLECTION 19,20 II. Specimens used in DNA testing Whole blood Solid tissue Serum and plasma Urine Bone marrow and many other like Hair

DNA STORAGE AND COLLECTION19,20:

10 DNA STORAGE AND COLLECTION 19,20 A. Blood and Bone Marrow -Collection tubes are EDTA or ACD -5-15 ml -Samples should not be frozen for transport -4-25°C

DNA STORAGE AND COLLECTION19,20:

11 DNA STORAGE AND COLLECTION 19,20 B. Serum Collection tubes with no additives 100 µl to 1 ml Transported at 20-25°C

DNA STORAGE AND COLLECTION19,20:

12 DNA STORAGE AND COLLECTION 19,20 C. Urine Urine container should be used for collection. At least 1 ml should be collected. Transported at 4-25°C

History/Background11:

13 History/Background 11 ‘ Southern’ hybridization named after Sir Edwin Southern Developed in 1975 Earned Sir Southern a Lasker Award in 2005

History/Background11:

14 History/Background 11 Spawned naming of related techniques: Southern blot (DNA) Northern blot (RNA) Western blot (Protein) Eastern blot (???)

Basic Techniques in Blotting13,14,15,11:

15 Basic Techniques in Blotting 13,14,15,11 Isolation of DNA, mRNA, Protein Purification of DNA, mRNA, Protein Gel Electrophoresis Transfer to Solid support Preparation of Probe Hybridization Washing Detection

1.Isolation 7,14:

16 1.Isolation 7,14 Breaking or opening of cell to expose Nucleic Acid- Bacterial Cell- By Combination of Enzymatic and Chemical treatment (Lysozyme + EDTA) Animal Cell- Cultured animal cell used, Direct treatment with detergent (SDS) Plant Cell- Strong cellulose cell wall, Cell are Frozen and then ground in mortar and pestle

2.Purification7:

17 2.Purification 7 DNA Purification -( Southern ) 1)Degradation of cellular component except DNA Cellular extract centrifuge supernatant Phenol treated aqueous layer treated with RNase RNA degraded DNA ppt. by adding Ethanol 2)Direct purification of DNA- Detergent CTAB added Insoluble complex of Nucleic acid ppt add in high salt sol n N.A. release treated with RNase RNA degraded DNA ppt. by adding Ethanol

2.Purification7:

18 3)Binding of DNA- Add silica particle in presence of Guanidium thiocynate followed by column chromatography DNA bind to silica 2.Purification 7

2.Purification7:

19 mRNA Purification -( Northern ) Cellular extract deproteinised by phenol/ chloroform mixture centrifuge aqueous phase ppt. by ethanol mRNA separated by affinity chromatography using Oligo (dt) –cellulose Protein Purification -( Western ) Using SDS (sodium dodecyl sulfate) detergent which coats the2’& 3’ protein structure with –ve charge 2.Purification 7

3.Gel Electrophoresis 1,3,4,12:

20 3.Gel Electrophoresis 1,3,4,12 In 1949 by chemist Linus Pauling . Electrophoresis used to separate individual according to size and charge or conformation. Gel electrophoresis used to separate plasma proteins for diagnostic purpose Polyacyamide or Agarose gels are used. Sample layered on matrix as a thin zone and then electrophoresed through matrix.

3.Gel Electrophoresis 1,3,4,12:

21 3.Gel Electrophoresis 1,3,4,12 Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.

3.Gel Electrophoresis 1,3,4,12,11,16:

22 3.Gel Electrophoresis 1,3,4,12,11,16 _ +

DNA Denaturation (Southern) 11:

23 T G A A T C A C A T T G DNA Denaturation ( Southern ) 11 Eliminate hydrogen bonds with sodium hydroxide (NaOH) or strong alkaline buffer

4. Transfer to solid support 1,3,4,12,11:

24 Two methods for transferring DNA to membrane -Capillary 4. Transfer to solid support 1,3,4,12,11

4. Transfer to solid support 1,3,4,12,11:

25 4. Transfer to solid support 1,3,4,12,11 - Electrophoretic

5.Preparation of probe 1, 12,11:

26 5.Preparation of probe 1, 12,11 Radioactive DNA probe for Southern & Northern -Restriction fragment of Plasmid containing gene of interest. -Plasmid is digested with particular restriction enzymes and digest is run on an Agarose gel. -Plasmid usually less than 20 kbp. -Template DNA is denatured by boiling. -DNA polymerase is added along with dATP, dGTP, radioactive dCTP

5.Preparation of probe 4,12,21:

27 Radioactive Antibodies for Western -Antibodies are raised by injecting a purified protein into animal (rabbit/mice) -Antibodies isolated by serum. -Labeled by chemically modifying side chain of tyrosine in the Antibody with iodine-125( 125 I ) Antibody-tyrosine + 125 I+H 2 O 2 H 2 O + 125iodine-tyrosine-antibody 5.Preparation of probe 4,12,21

* Pre-hybridization 21:

28 * Pre-hybridization 21 Pre-hybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane

6. Hybridization:

29 6. Hybridization 21

6. Hybridization:

30 6. Hybridization 21

7. Washes:

31 7. Washes 14,21

8.Detection:

32 8.Detection Autoradiography -It is a process of localization and recording of radiolabel with solid specimen -This produces image in photographic emulsion (silver halide crystal suspended in gelatin) -When Beta/Alpha ray from radiolabel passes through emulsion, silver ion converted into metallic silver atoms. -Development of visible image 7,14,21

8.Detection:

33 8.Detection Autoradiography 7,14,21

8.Detection:

34 8.Detection Autoradiography 7,14,21

8.Detection:

35 Enzymatic Development (Western) -In antibody-enzyme conjugate as a probe. -This can detected by soaking the filter in a solution of substrate for enzyme -This substrate produces an insoluble colored product (a chromogenic substrate) when acted upon by enzyme -This produces deposit of colored where the probe bound. 8.Detection 7,14,21

Summary :

36 Summary 21

Application:

37 Application Invaluable method of Gene analysis (Southern) Confirmation of DNA cloning result (Southern) Forensically applied to detect parenthood, rapist, thieves etc. (Southern,Northern) Tumor diagnosis In Infectious disease-for actual diagnosis of causative agent 22 In hereditary genetic disorder 9,16,15,22

Application:

38 Tissue transplantation HIV diagnosis (Western blot) 14 Bovine spongiform encephalopathy (BSE) commonly known as "mad cow disease” 22 Application 9,16,15

References:-:

39 References:- Victor L.Davdson,Donal B.Sittaman, Biochemistry, Lippincott Willams,Asia edition;4 th edition;2002: 22,195-198 Irfan A.Khan,Atiya Khanum, Recent Advances in Bioinformatics, Ukaaz Publication,1 st edition;2002: 52-55 Dr.Pradeep Parihar,A Textbook of basic an Molecular Genetics, Student edition,4 th edition; 2004: 265-266 Dr.SusanR.Barnus, Biotechnology, student publication, Asia edition;2006:49-67 Watson, Hopkins, Molecular Biology of Gene Benjamin Publication,4 th edition; 1987:608-609

References:-:

40 References:- Brian White MIT, Southern, Northern, Western & Cloning ‘Molecular searching’ techniques Page no: 1-32 V. Satyanarayana, U. Chakryapani, Biochemistry, Book’s and Allied pvt. Ltd ;3 rd edition; 2006:585-588 Ho Ming pang et al, High Throughput multiplexed electrophoresis in Drug Discovery, Drug Discovery Today,vol.9,No-4, Dec.2004:1072 Harsh Mohan, Textbook of Pathology, Jaypee Publication,5 th edition;2005:23,240 www.sceicherschuell.com/index.html

References:-:

41 References:- Ara H Der Marderosian, Biotechnology and drug’s, Remington ; Vol.1 st , 20 th edition ,2001:945 Dr.H.K.Das, Textbook of Biotechnology, Wiley Publication, 2 nd edition;2005:656,710,832.1041 Robert K.Murry, Harper’s Biotechnology, willey publication,24 th edition S.P.Vyas, N.K.Dixit, Pharmaceutical Biotechnology, CBS publication,1 st edition;1998:338,476 R.M.Twyman, Advanced Molecular Biology, Vivo Bo pvt. Ltd, 2 nd edition;2002:356-359

References:-:

42 References:- www.abitionthernacompany.com www.thefreedictonary.com www.molecularkitchen.com Dr.Dieter Weichenhan, collection and storage of Blood for DNA preparation, version 1.1, no.1.1, ( www.ngfn.com ) www.genetree.com Brian white, Southern, Northern, Western & Cloning: “Molecular searching” Technique, MIT, copyrigh1995 www.wikipediathefreeencyclopedia.com

Slide 43:

43 Thank you