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5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology1

WELCOME

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology2

PRESENTATION ON SYNTHESIS AND CLONING OF cDNA

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology3COURSE TITLE:- TECHNIQUES IN MOLECULAR BIOLOGY-2COURSE NO:-PBT 509(0+2)

COURSE TEACHER- Dr.K.M.Harini kumar PROFFESOR DEPT OF PLANT BIOTECHNOLOGYPRESENTED BY- AVINASH SHARMA I.D. NO- PALB3235 DEPT. OF PLANT BIOTECHNOLGY3

Contents:-Genomic libraries.Process of making cDNA and Genomic Libraries.Charactristics of cDNA.Synthesis of cDNA.Cloning of cDNA.Construction of cDNA Libraries.Advantages and Disadvantages of cDNA Libraries.Application of cDNA Libraries.Case study.Videos related to cDNA Libraries and Cloning.

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Genomic libraries: 5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology54/30/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology5Genomic 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 .

Genomic libraries cDNA libraries Gene library {made from genomic DNA}) {made from cDNA- copy of mRNA}

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology6cDNA libraries:-The cDNA library represents the population of mRNAs, it only contains the exons of proteins structural genes.mRNAReverse transcriptasecDNAreplicationdscDNAvectorrecombinate DNAE. colirecombinate DNA in E.coliProcess for making cDNA and Genomic libraries:-5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology7Tissue/CellmRNADNA cDNA (methylation; addition of linkers, etc.) Partial or Complete Restriction Vectors (cDNA: plasmids, phage Genomic: phage, cosmid, BAC, PAC, YAC, TAC)Size FractionationRestrictionLigationTransformation, in vitro packaging, etcScreening for desired clonesAmplify for long- term storage

Differences between cDNA AND Genomic Libraries:-5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology8Genomic LibrariescDNA Librariesfrom genomic DNA.reverse transcription of mRNA.

frequency of hits independent of gene expression levels.dependent.

may contain promoters and introns.no promoters or introns.

cannot express in heterologous system.expression is feasible if linked to a suitable promoter.useful for genome analysis, map-based cloning, promoter studies, etc.useful for analysis of coding regions and gene functions.Characteristics of cDNA libraries:-

Reverse transcription of mRNADependent on gene expressionNo intronsExpression is feasible if linked to a suitable promoterUseful for analysis of coding regions and gene functionsNo cDNA library was made from prokaryotic mRNA because prokaryotic 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.

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Synthesis of cDNA:-

Synthesis of cDNA will be done through:- a}Self primer methodb}Cloning vectors

Mechanism of synthesis of cDNA:-mRNA isolation, purificationCheck the RNA integritySynthesis of cDNA Treatment of cDNA endsLigation to vector

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology10mRNA 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.AAAAAAAAAAn5 cap5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology115/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology12Methods:Translating the mRNA : use cell-free translation system if the mRNAs can be translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gelsEnrichment:- carried out by hybridization

Check the mRNA integrityAngelia 0912Make sure that the mRNA is not degraded. 5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology13Synthesis of cDNA:-A short oligo(dT) primer is used. It anneals to the mRNAs poly(A) tail, allows reverse transcriptase to synthesize the cDNA (DNA-mRNA hybrid).Rnase H degrades the mRNA strand, creating small fragments that serve as primersDNA polymerase I makes new DNA fragments, DNA ligase connects them to make a complete chain.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology14cDNA from a Polyadenylated mRNAcDNA: first strand synthesisProduct is complementary DNA, called cDNA. It is equivalent to the template strand of the duplex DNA.AAAAAAA53mRNATTTTTAAAAAAA53TTTTTAnneal oligo-dT primerReverse transcriptase:RNA-directed DNA polymeraseRNase HdNTPsTTTTTAAAAAAA53Hydrolyze remaining RNA with baseTTTTT55/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology15cDNA: second strand synthesisTTTTT5cDNA Problem: How to get a primer for 2nd strand synthesis?TTTTT5dCTPs Terminal deoxynucleotidyl transferase CCCCTTTTT5CCCCGGGGGGGG Ligate an adaptor to the 3 end5353TTTTT5CCCCGGGG53DNA polymeraseAAAAAdNTPsDuplex cDNA35/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology16

Cloning of cDNAVectors for cDNA Libraries:- Vectors Insert Size Remarks PhagesUp to 20-30 kb(for replacement vectors) and 10-15 kb (for insertion vectors)Maximum size for mRNA is about 8 kb, hence the capacity of DNA insert is not a major concern here. Insertion vector system is usually employed. Useful for study of individual genes and their putative functions. Efficient packaging system, easy for gene transfer into E. coli cells, more representative than plasmid libraries, subcloning and subsequent DNA manipulation processes are less convenient than plasmid system.Bacterial plasmidsUp to 10-15 kbRelatively easy to transform E. coli cells although may not be as efficient as the phage system for large scale gene transfer. Less representative than phage libraries, sub cloningand subsequent DNA manipulation processes are more convenient than the phage systems.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology175/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology18cDNA Cloning:-Introduction of restriction site linkers to the ends of the cDNA by blunt end ligation.Digestion with restriction enzyme to create sticky ends.Mixing cDNA with vector DNA cut with the same restriction enzyme in the presence of DNA ligaseTransforming into an E. coli host for cloning.Use linkers engineered with appropriate ssDNA overhangs so do not digest.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology19Linkers 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.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology20cDNA Libraries:-A large number of clones, often pooled together (so you have to fish out the one you want), but sometimes ordered. Genomic library vs. cDNA. Genomic uses enough input DNA to cover the genome 5-10x, so chance fluctuations don't prevent all sequences from being cloned. Repeat sequence DNA is a problem. cDNA libraries are usually made from single tissues: expression varies between tissues. Large difference in expression levels, often compensated for by normalizing the library: trying to equalize copy number of different sequences. Detection of clones containing specific genes is generally by hybridization with labeled probes. It can also be done using antibodies if the genes in the library are being expressed.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology21Construction cDNA libraries:-

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5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology23Hybridization:-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 gelNorthern blot: uses RNA on the gel instead of DNAin situ hybridization: probing a tissueColony hybridization: detection of clonesMicroarraysPolymerase Chain Reaction5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology24Labeling:-Several methods. One is random primers labeling: use 32P-labeled dNTPsshort random oligonucleotides as primers (made synthetically) single stranded DNA template (made by melting double stranded DNA by boiling it)DNA polymerase copies the DNA template, making a new strand that incorporates the label. Can also label RNA (sometimes called riboprobes), use non-radioactive labels (often a small molecule that labeled antibodies bind to, or a fluorescent tag), use other labeling methods.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology25Hybridization Process:-All the DNA must be single stranded (melt at high temp or with NaOH). Occurs in a high salt solution at say 60oC. Complementary DNAs find each other and stick. Need to wash off non-specific binding. Stringency: how perfectly do the DNA strands have to match in order to stick together? Less than perfect matches will occur at lower stringency (e.g. between species). Increase stringency by increasing temp and decreasing salt concentration. Rate of hybridization depends on DNA concentration and time (Cot), as well as GC content and DNA strand length.Autoradiography. Put the labeled DNA next to X-ray film; the radiation fogs the film.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology26Southern Blot:-Used to detect a specific DNA sequence in a complex mixture, such as genomic DNACut 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 heatHybridize membrane to a radioactive probe, then detect specific bands with autoradiography.

Northern blot uses RNA instead of DNA. RNA must be denatured so the distance it migrates on the gel is proportional to its length: put formaldehyde in the gel.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology27Colony Hybridization:-

Bacterial colonies (or phage plaques) containing recombinant DNA are grown on agar, then blotted to nitrocellulose and hybridized as with Southern blots.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. 5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology28In Situ Hybridization:-Using tissues or tissue sections.Often done with non-radioactive probes because the high energy of 32P emission gives an imprecise view of hybridization.Counterstain the tissue so non-hybridizing parts are visible.

285/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology29Microarrays:-Place probes from many different genes on a glass microscope slide, then hybridize to cDNA made from messenger RNA isolated from a tissue. You 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 labeledOften 2 conditions are compared (control and experimental), using red and green fluorescent tags.Semi-quantitative

Can also be used to screen for DNA mutations.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology30Polymerase Chain Reaction:-Another way to get your gene and it is very common.Based on a knowledge of the DNA sequence of a piece of DNA.Allows you to design primers that, along with a thermostable DNA polymerase, lets you make all the DNA that you need.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology31

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Advantages of cDNA:- cDNA library is a collection of actively expressed genes in the cells or tissue from which the mRNA was isolated.You just 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.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology34Disadvantages 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.You 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.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology35Application 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.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology365/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology37Case studyResearch Title:- High Occurrence of Functional New Chimeric Genes in Survey of Rice Chromosome 3 Short Arm Genome Sequences.Chengjun Zhang, Jun Wang, Nicholas C. Marowsky, Manyuan Long, Rod A. Wing, and Chuanzhu Fan. Published:- 7 May , 2013In an effort to identify new genes in rice, we searched the genomes of Asian-cultivated rice Oryza sativa ssp. japonica and its wild progenitors, looking for lineage-specific genes. Using genome pairwise comparison of approximately 20-Mb DNA sequences from the chromosome 3 short arm (Chr3s) in six rice species, O. sativa, O. nivara, O. rufipogon, O. glaberrima, O. barthii, and O. punctata, combined with synonymous substitution rate tests and other evidence, we were able to identify potential recently duplicated genes, which evolved within the last 1Myr.Results:- He identified that 28 functional O. sativa genes, which likely originated from O. glaberrima. These genes account for around 1% (28/3,176) of all annotated genes on O. sativas Chr3s.Among the 28 new genes, two duplicated segments contained eight genes. Fourteen of the 28 new genes consist of chimeric gene structure derived from one or multiple parental genes and flanking targeting sequences. Although the majority of these 28new genes were formed by single or segmental DNA-based gene duplication and recombination, we found two genes that were likely originated partially through exon shuffling. We showed all 28 new genes appeared to be functional, as suggested by the presence of RNA-sequence, cDNA, expressed sequence tag, massively parallel signature sequencing, and/or small RNA data.

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Research title:- Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici)Peng Ling, Meinan Wang, Xianming Chen and Kimberly Garland Campbell.Published: 4 June 2007.

Background:- Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi.Results:- A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using uredospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%). In addition, 36 clones (18.4%) had significant homology to hypothetical proteins, 37 clones (18.9%) had some homology to genes in other fungi, and the remaining 50 clones (25.5%) did not produce any hits. Conclusion:- The full-length cDNA library is useful in identifying functional genes of P. striiformis

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Reasearch title:- Cloning and characterization of two Argonaute genes in wheat (Triticum aestivum L.)Fanrong Meng, Haiying Jia, Na Ling, Yinlei Xue, Hao Liu, Ketao Wang, Jun Yin and Yongchun Li.Accepted 18 February 2013

Background:- Argonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing. Various classes of Argonaute genes have been identified from plants and might be involved in developmental regulation. However, these genes found in wheat (Triticum aestivum).Results:- In this study, two full-length cDNAs of Argonaute proteins were cloned from wheat, designated as TaAGO1b and TaAGO4. The cDNA of TaAGO1b is 3273 bp long and encodes 868 amino acids, with a molecular weight of ~97.78 kDa and the 3157-bp TaAGO4 encodes 916 amino acids, with a molecular mass of 102.10 kDa . Genomics analysis showed that TaAGO1b and TaAGO4 contain 20 and 18 introns, respectively. Protein structural analysis demonstrated that typical PAZ and PIWI domains were found in both TaAGO1b and TaAGO4. Highly conserved PIWI domains, we detected conserved Asp-Asp-His (DDH) motifs that function and critical roles during the process of sequence-specific cleavage in the RNAi machinery.. 5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology40Continued:- Structural modelling indicated that both TaAGOs can fold to a specific / structure. Moreover, the three aligned DDH residues are spatially close to each other at the slicer site of the PIWI domain. Expression analysis indicated that both genes are ubiquitously expressed in vegetative and reproductive organs, including the root, stem, leaf, anther, ovule, and seed. However, they are differentially expressed in germinating endosperm tissues. This two TaAGOs are also differentially expressed in developing wheat plants and that their expression patterns are affected by vernalization treatment.

Conclusions:-Two wheat Argonaute genes, TaAGO1b and TaAGO4, were cloned. Phylogenetic analysis, prediction of conserved domains and catalytic motifs, and modelling of their protein structures suggested that they encode functional Argonaute proteins. Temporal and spatial expression analyses indicated that these genes are potentially involved in developmental regulation of wheat plants.5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology41

Rearch title:- Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheatShanjun Tian, Xinguo Mao, Hongying Zhang, Shuangshuang Chen, Chaochao Zhai, Shimin Yang and Ruilian JingPublished:- 11 March 2013Environmental stresses such as drought, salinity, and cold are major adverse factors that significantly affect agricultural productivity. Protein phosphorylation/dephosphorylation is a major signalling to induced the osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) family members play essential roles in the response to hyperosmotic stresses in plants. TaSnRK2.3 gene, a novel SnRK2 member was cloned, and three copies located on chromosomes 1A, 1B, and 1D in common wheat. TaSnRK2.3 was strongly expressed in leaves, and responded to polyethylene glycol, NaCl, abscisic acid, and cold stresses. To characterize its function, transgenic Arabidopsis overexpressing TaSnRK2.3GFP controlled by the cauliflower mosaic virus 35S promoter was generated and works s in severe abiotic stresses. Results::-Overexpression of TaSnRK2.3 resulted in an improved root system and significantly enhanced tolerance to drought, salt, and freezing stresses, simultaneously it enhanced expression of abiotic stress-responsive genes and ameliorative physiological indices, including a decreased rate of water loss, enhanced cell membrane stability, improved photosynthetic potential, and significantly increased osmotic potential and free proline content under normal and/or stressed conditions. It also determine that TaSnRK2.3 is a multifunctional regulator, with potential for utilization in transgenic breeding for improved abiotic stress tolerance in crop plants.

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology42 Videos of cDNA Synthesis:- VIDEO-1

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology43 cDNA Synthesis:- VIDEO-2

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology44 Cloning of cDNA:- VIDEO-1

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology45 Cloning of cDNA:- VIDEO-2

5/26/2014PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology46References:-Construction and Screening of Gene Libraries Genome IIby TA Brown.Recombinant DNA cloning technology by Gene Libraries Genome IIby TA Brown. High Occurrence of Functional New Chimeric Genes in Survey of Rice Chromosome 3 Short Arm Genome Sequences by Chengjun Zhang, Jun Wang, Nicholas C. Marowsky, Manyuan Long, Rod A. Wing, and Chuanzhu Fan. Genome Biol. Evol.Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) by Peng Ling, Meinan Wang, Xianming Chen and Kimberly Garland Campbell. BioMed Central. 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 commonwheat by ShanjunTian, XinguoMao, HongyingZhang, ShuangshuangChen,ChaochaoZhai, ShiminYang and RuilianJing. Journal of Experimental Botany.Videos:- Jove.com, TNAU{CPMB} and Youtube.

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