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5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
1
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
2
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
3
COURSE TITLE:- TECHNIQUES IN MOL...
Contents:-
• Genomic libraries.
• Process of making cDNA and Genomic Libraries.
• Charactristics of cDNA.
• Synthesis of c...
Genomic libraries:—
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
54/30/2014
PBT...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
6
cDNA libraries:-
The cDNA librar...
Process for making cDNA and Genomic libraries:-
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant ...
Differences between cDNA AND Genomic Libraries:-
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant...
Characteristics of cDNA libraries:-
• Reverse transcription of mRNA
• Dependent on gene expression
• No introns
• Expressi...
Synthesis of cDNA:-
Synthesis of cDNA will be done through:-
a}Self primer method
b}Cloning vectors
Mechanism of synthesis...
mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
• oligo (dT) can be bound to the poly(A) tail
a...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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Methods:
Translating the mRNA :...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
13
Synthesis of cDNA:-
•A short ol...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
14
cDNA from a Polyadenylated mRNA...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
15
cDNA: second strand synthesis
T...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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Cloning of cDNA
Vectors for cDNA Libraries:-
Vectors Insert Size Remarks
λ Phages Up to 20-30 kb(for replacement
vectors) and 10-15 kb (fo...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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cDNA Cloning:-
•Introduction of...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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Linkers for Cloning DNA:-
• Any...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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cDNA Libraries:-
• A large numb...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
21
Construction cDNA libraries:-
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
22
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
23
Hybridization:-
• The idea is t...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
24
Labeling:-
• Several methods. O...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
25
Hybridization Process:-
• All t...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
26
Southern Blot:-
• Used to detec...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
27
Colony Hybridization:-
•Bacteri...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
28
In Situ Hybridization:-
• Using...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
29
Microarrays:-
• Place probes fr...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
30
Polymerase Chain Reaction:-
• A...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
31
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
32
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
33
Advantages of cDNA:-
• cDNA library is a collection of actively expressed genes
in the cells or tissue from which the mRNA...
Disadvantages of cDNA:-
• One disadvantage is that cDNA libraries can be difficult
to create and screen if a source tissue...
Application of cDNA library:-
• Discovery of novel genes.
• Elucidation of gene function.
• In vitro study of gene functio...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
37
Case study
Research Title:- High Occurrence of Functional New Chimeric Genes in Survey
of Rice Chromosome 3 Short Arm Genome Sequence...
Research title:- Construction and characterization of a full-length cDNA
library for the wheat stripe rust pathogen (Pucci...
Reasearch title:- Cloning and characterization of two Argonaute genes in wheat
(Triticum aestivum L.)
Fanrong Meng, Haiyin...
Continued:-
• Structural modelling indicated that both TaAGOs can fold to a specific α/β
structure. Moreover, the three al...
Rearch title:- Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common
wheat
Shanjun Tian, Xinguo Mao, Hon...
Videos of cDNA Synthesis:-
VIDEO-1
5/26/2014 PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology...
cDNA Synthesis:-
VIDEO-2
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
44
Cloning of cDNA:-
VIDEO-1
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
45
Cloning of cDNA:-
VIDEO-2
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
46
References:-
• Construction and Screening of Gene Libraries “Genome II”by TA Brown.
• Recombinant DNA cloning technology b...
5/26/2014
PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
48
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Avinash ppt

  1. 1. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 1
  2. 2. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 2
  3. 3. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 3 COURSE TITLE:- TECHNIQUES IN MOLECULAR BIOLOGY-2 COURSE NO:-PBT 509(0+2) COURSE TEACHER- Dr.K.M.Harini kumar PROFFESOR DEPT OF PLANT BIOTECHNOLOGY PRESENTED BY- AVINASH SHARMA I.D. NO- PALB3235 DEPT. OF PLANT BIOTECHNOLGY 3
  4. 4. 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 4
  5. 5. Genomic libraries:— 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 54/30/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 5 • Genomic 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}
  6. 6. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 6 cDNA libraries:- The cDNA library represents the population of mRNAs, it only contains the exons of protein’s structural genes. mRNA Reverse transcriptas cDNA replication dscDNA vector recombinate DNA E. coli recombinate DNA in E.coli
  7. 7. Process for making cDNA and Genomic libraries:- 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 7 Tissue/Cell mRNA DNA cDNA (methylation; addition of linkers, etc.) Partial or Complete Restriction Vectors (cDNA: plasmids, λphage Genomic: λphage, cosmid, BAC, PAC, YAC, TAC) Size Fractionation Restriction Ligation Transformation, in vitro packaging, etc Screening for desired clones Amplify for long- term storage
  8. 8. Differences between cDNA AND Genomic Libraries:- 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 8 Genomic Libraries cDNA Libraries •from 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.
  9. 9. Characteristics of cDNA libraries:- • Reverse transcription of mRNA • 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 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 9
  10. 10. Synthesis of cDNA:- Synthesis of cDNA will be done through:- a}Self primer method b}Cloning vectors Mechanism of synthesis of cDNA:- • mRNA isolation, purification • Check the RNA integrity • Synthesis of cDNA • Treatment of cDNA ends • Ligation to vector 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 10
  11. 11. 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. AAAAAAAAAAn5’ cap 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 11
  12. 12. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 12 Methods: Translating the mRNA : use cell-free translation system if the mRNAs can be translated Analysis 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 gels Enrichment:- carried out by hybridization Check the mRNA integrity Angelia 09 12 Make sure that the mRNA is not degraded.
  13. 13. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 13 Synthesis of cDNA:- •A short oligo(dT) primer is used. It anneals to the mRNA’s 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 primers •DNA polymerase I makes new DNA fragments, DNA ligase connects them to make a complete chain.
  14. 14. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 14 cDNA from a Polyadenylated mRNA cDNA: first strand synthesis Product is complementary DNA, called cDNA. It is equivalent to the template strand of the duplex DNA. AAAAAAA5’ 3’mRNA TTTTT AAAAAAA 5’ 3’ TTTTTAnneal oligo-dT primer Reverse transcriptase: RNA-directed DNA polymerase RNase H dNTPs TTTTT AAAAAAA 5’ 3’ Hydrolyze remaining RNA with base TTTTT 5’
  15. 15. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 15 cDNA: second strand synthesis TTTTT 5’cDNA Problem: How to get a primer for 2nd strand synthesis? TTTTT 5’ dCTPsTerminal deoxynucleotidyl transferase CCCC TTTTT 5’CCCC GGGG GGGG Ligate an adaptor to the 3’ end 5’ 3’ 5’ 3’ TTTTT 5’CCCC GGGG5’ 3’ DNA polymerase AAAAA dNTPs Duplex cDNA 3’
  16. 16. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 16 Cloning of cDNA
  17. 17. Vectors for cDNA Libraries:- Vectors Insert Size Remarks λ Phages Up 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 plasmids Up to 10-15 kb • Relatively easy to transform E. coli cells although may not be as efficient as the λphage system for large scale gene transfer. • Less representative than λphage libraries, sub cloningand subsequent DNA manipulation processes are more convenient than the λphage systems. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 17
  18. 18. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 18 cDNA 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 ligase •Transforming into an E. coli host for cloning. • Use linkers engineered with appropriate ssDNA overhangs so do not digest.
  19. 19. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 19 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.
  20. 20. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 20 cDNA 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.
  21. 21. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 21 Construction cDNA libraries:-
  22. 22. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 22
  23. 23. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 23 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 • in situ hybridization: probing a tissue • Colony hybridization: detection of clones • Microarrays • Polymerase Chain Reaction
  24. 24. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 24 Labeling:- • Several methods. One is random primers labeling: • use 32P-labeled dNTPs • short 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.
  25. 25. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 25 Hybridization 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.
  26. 26. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 26 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. • 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.
  27. 27. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 27 Colony 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.
  28. 28. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 28 In 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.
  29. 29. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 29 Microarrays:- • 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 labeled • Often 2 conditions are compared (control and experimental), using red and green fluorescent tags. • Semi-quantitative • Can also be used to screen for DNA mutations.
  30. 30. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 30 Polymerase 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, let’s you make all the DNA that you need.
  31. 31. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 31
  32. 32. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 32
  33. 33. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 33
  34. 34. 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 34
  35. 35. 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. • 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 35
  36. 36. 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 36
  37. 37. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 37 Case study
  38. 38. Research 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 , 2013 • In 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. sativa’s 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 38
  39. 39. 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 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 39
  40. 40. 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. . 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 40
  41. 41. Continued:- • Structural modelling indicated that both TaAGOs can fold to a specific α/β 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 41
  42. 42. Rearch title:- Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat Shanjun Tian, Xinguo Mao, Hongying Zhang, Shuangshuang Chen, Chaochao Zhai, Shimin Yang and Ruilian Jing Published:- 11 March 2013 • Environmental 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 TaSnRK2.3–GFP 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. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 42
  43. 43. Videos of cDNA Synthesis:- VIDEO-1 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 43
  44. 44. cDNA Synthesis:- VIDEO-2 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 44
  45. 45. Cloning of cDNA:- VIDEO-1 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 45
  46. 46. Cloning of cDNA:- VIDEO-2 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 46
  47. 47. References:- • Construction and Screening of Gene Libraries “Genome II”by TA Brown. • Recombinant DNA cloning technology by Gene Libraries “Genome II”by 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 common wheat by Shanjun Tian, Xinguo Mao, Hongying Zhang, Shuangshuang Chen,Chaochao Zhai, Shimin Yang and Ruilian Jing. ” Journal of Experimental Botany”. • Videos:- Jove.com, TNAU{CPMB} and Youtube. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 47
  48. 48. 5/26/2014 PBT 505 Techniques in Molecular Biology-2 {0+2} Deptt. of Plant Biotechnology 48

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