Synthesis and cloning of c dna

synthesis of cDNA
Cloning of cDNA

1. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 { + 2} D e p t t. o f Pla n t B iote c hn ol o gy 1 WELCOME 1

2. COURSE TITLE :- TECHNIQUES IN MOLECULA R BIOLOGY-2 COURSE NO:-PBT 509(0 +2 ) TOPIC: SYNTHESIS AND CLONING OF cDNA COURSE TEACHER- Dr.K.M.Harini kumar PROFFESOR DEPT OF PLANT BIOTECHNOLOGY PRESENTED BY- BHARATI.G.S. I.D. NO- PAL B9299 DEPT. OF PLANT BIOTECHNOLGY UAS,GKVK,BANGLORE. 2

3. Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis. Gene library Genomic libraries (made from genomic DNA) cDNA libraries (made from cDNA- copy of mRNA I1 Genomic libraries 3

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5. What is cDNA library ? cDNAlibrary is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. 5

6. cDNA libraries:- The cDNA library represents the population of mRNAs, it only contains the exons of protein’s structural genes. mRNA Reverse transcripta cDNA replicati o n dscDNA ve c tor recombinate DNA E. coli recombinate DNA in E.coli 6

7. Importance of cDNA libraries 1. No cDNA library was made from prokaryotic mRNA . • • Prokaryotic mRNA is very unstable Genomic libraries of prokaryotes are easier to make and contain all the genome sequences. 7

8. 2) cDNA libraries are very useful for eukaryotic gene analysis Condensed protein encoded, gene libraries ,have much less junk sequences. cDNAs have no introns so genes can be expressed in E.coli directly. Are very useful to identify new genes. 8

9. Construction cDNA libraries:- 9

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12. Characteristics of cDNA libraries:- Reverse transcription of mRN A Dependent on gene expression No introns Expression is feasible if linked to a suitable promoter Useful for analysis of coding regions and gene functions No cDNA library was made from prokaryotic mRNA because prokaryotic . 12

13. mRNA are very unstable and easy to make genomic library. cDNA libraries are very useful for eukaryotic gene analysis because condensed protein encoded gene library have much less junk sequences. Very useful to identify genes. Tissue or cell specific. 13

14. mRNA isolation, purification Check theRNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector 14 Synthesis of cDNA :-

15. mRNA isolation Most eukaryotic mRNAs are polyadenylated at their 3’ ends oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA. AAAAAAAAAAn 5’ cap 15

16. Three methods to isolate mRNA. Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet Alternative route of isolating mRNA is lysing cells and then preparing mRNA- ribosome complexes on sucrose gradients 16

17. Make sure that the mRNA is not degraded. Methods : Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated Analysis the mRNAs by gel elctrophoresis : use agarose or polyacrylamide gels Check the mRNA integrity 17

18. Cloning the particular mRNAs Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library. Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gels Enrichment: carried out by hybridization Example: clone the hormone induced mRNAs (substrated cDNA library) 18

19. Synthesis of cDNA : First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’- end of the first strand and then use a complementary primer to make the second. 19

20. 5 ’ m RNA AA A A A - 3 ’ HO- TTTTT P -5’ Reverse transcriptase Four dNTPs TTTTT P -5’ 5’ mRNA AAAAA -3’ c D NA c D NA c D NA TTTTT P -5’ 3 ’ 3’-CCCCCCC TTTTT P -5’ Terminal transferase Alkali (hydrolyaes RNA) Purify DNA oligo(dG) Klenow polymerase or reverse Transcriotase Four dNTPs dCTP 5’ mRNA AAAAA -3’ 5’-pGGGG-OH 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC TTTTT P -5’ - 3 ’ Duplex c DNA The first strand synthesis 20

21. 5 ’- p GG G G 3 ’-C CCCC CC 3’-GGCTTAAGCCCCCC 5’-pAATTCGGGGGG TTTTTGGCTTAAGCC-OH CC GA A T T C GG- 3 ’ 3 ’- CCC C 3’-CCCCCCC 5’-pGGGG 5’-pGGGG T T TT Tp -5’ - 3 ’ T T TT Tp -5’ - 3 ’ - 3 ’ TTTTTGGCTTAAp-5’ HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH CC G - 3 ’ Duplex cDNA Single strand-specific nuclease 3 ’ - CC C TT T T T p - 5’ Klenow polymerase treat with E.coRI methylase Add E.colRI linkers using T4 DNA ligase HO-CCGAATTCGGGGGG E.colRI digestion Ligate to vector and transfom Second strand synthesis 21

22. Treatment of cDNA ends Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning. The process : Move protruding 3’-ends (strand-special nuclease) Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs) Ligate the blunt-end and linkers (T4 DNA ligase) Restriction enzyme digestion (E.coRI ) Tailing with terminal transferase or using adaptor molecules 22

23. Ligation to vector Any vectors with an E.coRI site would suitable for cloning the cDNA. The process : Dephosphorylate the vector with alkaline phosphatase Ligate vector and cDNA with T4 DNA ligase (plasmid or λ phage vector) 23

24. Vectors According to genome’s size,we can select a proper vector to construct a library . V e cto r s P l a s m i d i n s e r t (k b ) 5 p ha g e λ 23 cosmid YAC 4 5 1 00 The most commonly chosen genomic cloning vectors are λ relacement vectors which must be digested with restriction enzymes to produce the two λ end fragment or λ arms between which the genomic DNA will be digested I1 Genomic libraries 24

25. c o s c o s Long (left) arm short (right) a r m Exogenous DNA (~20-23 kb) λ phage vector in cloning c o s c o s Long (left) arm short (right) arm Exogenous DNA (~20-23 kb) 25

26. Linkers for Cloning DNA Any DNA fragment can have a specific restriction site added to the ends by ligating on a “linker”. Linkers are small, synthetic (made in the lab, or ordered from a company) DNA fragments which contain the recognition sequence for one or more restriction enzymes. After ligating on linkers, the DNA is cut with the appropriate restriction enzyme to produce ends for cloning . 26

27. Hybridization:- The idea is that if DNA is made single stranded (melted), it will pair up with another DNA (or RNA) with the complementary sequence. If one of the DNA molecules is labeled, you can detect the hybridization. Basic applications: Southern blot : DNA digested by a restriction enzyme then separated on an electrophoresis gel Northern blot : uses RNA on the gel instead of DNA Colony hybridization : detection of clones Microarrays Polymerase Chain Reaction 27

28. Southern Blot:- Used to detect a specific DNA sequence in a complex mixture, such as genomic DNA Cut DNA with restriction enzyme, then run on an electrophoresis gel. Suck buffer through the gel into a nitrocellulose membrane. The buffer goes through but the DNA sticks to the membrane. Fix the DNA to the membrane permanently with UV or heat Hybridize membrane to a radioactive probe, then detect specific bands with autoradiography. 28

29. Colony Hybridization:- Bacterial colonies (or phage plaques) containing recombinant DNA are grown on agar, then blotted to nitrocellulose and hybridized as with probe. The colonies on the agar plates stay alive, and once the correct colony has been detected, it can be picked and grown up for further work. 29

30. 28 In Situ Hybridization:- Using tissues or tissue sections. Often done with non- radioactive probes because the high energy of 32 P emission gives an imprecise view of hybridization. Counterstain the tissue so non-hybridizing parts are visible. 30

31. Microarrays:- Place probes from many different genes on a glass microscope slide, then hybridize to cDNA made from messenger RNA isolated from a tissue . we see which genes are active in that tissue. Mostly done with 60mers: 60 bases long, synthetic oligonucleotides, made using sequence information from the genes. cDNA is fluorescently labeled Often 2 conditions are compared (control and experimental), using red and green fluorescent tags . 31

32. Polymerase Chain Reaction:- 32 Another way to get our gene and it is very common. Based on a knowledge of the DNA sequence of a piece of DNA. Allows we have to design primers that, along with a thermostable DNA polymerase, let’s make all the DNA that we need.

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36. Advantages of cDNA:- cDNA library is a collection of actively expressed genes in the cells or tissue from which the mRNA was isolated. We will get the expressed genes. Introns are not cloned in a cDNA library, which greatly reduces the total amount of DNA that is cloned compared to a genomic library. it isolate homologous genes. cDNA of proteins can facilitate to generate antibodies and monoclonal antibodies. The most important application of cDNA library is to study expression of mRNA. 36

37. Disadvantages of cDNA:- One disadvantage is that cDNA libraries can be difficult to create and screen if a source tissue with an abundant amount of mRNA for the gene is not available. we don't get control sequences or introns, and frequency depends on level of expression. First strand synthesis often does not go to completion. Individual cDNA clones will frequently have the reverse complement of only part of the mRNA. Multiple cDNA clones from a single mRNA will be present in the library. Priming second strand synthesis is inefficient. 37

38. Application of cDNA library:- Discovery of novel genes. Elucidation of gene function. In vitro study of gene function. To obtain pure sample of a gene. To get high yields of recombinant cDNA . Commercial production of proteins and other biological molecules. Study the alternative splicing. Carcinogen identification. 38

39. REFERENCES Biotechnology expanding horizon by B.D.Singh Biotechnology by P.K.Gupta Cloning and characterization of two Argonaute genes in wheat ( Triticum aestivum L.) by Fanrong Meng , Haiying Jia , Na Ling, Yinlei Xue , Hao Liu, Ketao Wang, Jun Yin and Yongchun . “ BioMed Central “. Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat by Shanjun Tian , Xinguo Mao, Hongying Zhang, Shuangshuang Chen,Chaochao Zhai , Shimin Yang and Ruilian Jing. ” Journal of Experimental Botony ”. 39

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