SOURCE ORGANISM GENE CLONING GENE CLONING ISOLATION OF PURIFIED DNA ?GENERATION OF FRAGMENTS WITH THE GENE OF INTEREST SELECTION OF VECTOR PREPARATION OF VECTOR AND INSERT WITH COMPATIBLE ENDS LIGATION AND RECOMBINANT MOLECULE INTRODUCTION OF RECOMBINANT DNA INTO THE CELL SCREENING AND SELECTION OF THE DESIRED RECOMBINANT DNA MODIFYING ENZYMES NUCLEASES, PHOSPHATASES, KINASES ETC. Plasmid based vectors Phage based vectors Yeast expression vectors
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SOURCE ORGANISMGENE CLONINGGENE CLONING
ISOLATION OF PURIFIED DNA
?GENERATION OF FRAGMENTS WITH THE GENE OF INTEREST
SELECTION OF VECTOR
PREPARATION OF VECTOR AND INSERTWITH COMPATIBLE ENDS
LIGATION AND RECOMBINANT MOLECULE
INTRODUCTION OF RECOMBINANT DNA INTO THE CELL
SCREENING AND SELECTION OF THE DESIRED RECOMBINANT
DNA MODIFYING ENZYMES NUCLEASES, PHOSPHATASES, KINASES ETC.
Plasmid based vectorsPhage based vectorsYeast expression vectors
SCREENING AND SELECTION OF THE DESIRED RECOMBINANT
TransformationTransfection
Selection for recombinantsSelection of desired clone
Selection of desired clone
Direct Selection for the desired gene
Identification of the clone from a gene library
Direct selection by additional phenotype
Direct selection by marker rescue
Genomic and cDNA library
A genomic library is a collection of clones sufficient in number to contain every single gene present in a particular organism
N= -------------
ln(1-P)
ln(1-a/b)
N= No. of clones; P= probability of finding a gene; a= average insert size; b = total size of the genome
Synthesis & cloning of cDNA
cDN
A li
brar
y
Colony hybridization
Labelling DNA by Random priming
Non-radioactive methods of DNA labelling
Labelling with biotinylation
• Biotinylated probes are prepared through a nick-, translation reaction by replacing nucleotides with biotinylated derivatives. After hybridization and washing, detection of hybrids is done by a series of cytochemical reactions which finally give a blue colour whose intensity is proportional to the amount of biotin in the hybrid.
• There are several advantages of using biotinylated probes. For example, these assays employ non toxic materials, whose half-life is longer. These probes can be prepared in advance in bulk and stored at - 20°C for repeated uses. Detection of hybrids is much faster than by radioactive probes.
Screening based on abundance eg. Wheat gliadin cDNA
Sources of probes and primers
Similarity to known genes
Homology cloning (mouse clone used to obtain human gene)
Have idea about Protein sequence
Complementary genetics • predicting gene sequence from protein• antibody based immunoscreening
Chromosomal location
Map-based cloning (using genetic approach)Wait till you are taught Molecular markers
Cloning of genes based on defferential expression patterns
DDPCRSSH (suppression subtractive hybridization)
Complementary Genetics
1. Protein sequence is related to gene sequence
NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-
ATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G
2. The genetic code information is used to design PCR primers
Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence
a. template DNA is melted (94C)
3’ 5’5’ 3’
3’ 5’
5’ 3’
b. primers anneal to complementary site in melted DNA (55C)
3’ 5’
5’ 3’
3’ 5’
5’ 3’
c. two copies of the template DNA made (72C)
Complementary Genetics(cont.)
Suppression subtractive hybridization (SSH; Diatchenko et al. 1996).
mRNA differentially display polymerase chain reaction (DD PCR; Liang et al 1992)
Representational difference analysis (RDA; Nikolai et al.1993)
Microarray (Chee et al. 1996)
Methods for discovering differentially expressed genes
Suppression subtractive hybridization
(SSH)
♠ A powerful technique for identification of differential gene expression
♠ SSH is used to selectively amplify target cDNA fragments (differentially expressed) and simultaneously suppress non target DNA amplification.
♠ Rare differentially expressed genes can be enriched by 1000-5000 folds
Principle :
It is based on suppression PCR and hybridization.
TechniTechniques ques
AdvantageAdvantage DisadvantageDisadvantage
SSHSSH 1. Relatively smaller 1. Relatively smaller quantities of samples;quantities of samples;
2. High efficacy, 2. High efficacy, especially efficient forespecially efficient for
can be obtained in can be obtained in resultant libraryresultant library
1. Low sensitivity;1. Low sensitivity;2. Largest quantities 2. Largest quantities
of samplesof samples3. Labor intensive3. Labor intensive
Advantage and disadvantage of gene expression profile
Suppression subtractive hybridization
(SSH)
♠ A powerful technique for identification of differential gene expression
♠ SSH is used to selectively amplify target cDNA fragments (differentially expressed) and simultaneously suppress non target DNA amplification.
♠ Rare differentially expressed genes can be enriched by 1000-5000 folds
Principle :
It is based on suppression PCR and hybridization.
The method
♦ Synthesis of tester/driver Synthesis of tester/driver cDNAs cDNAs
♦ Digestion by a four base Digestion by a four base cutting restrictive enzyme cutting restrictive enzyme
♦ Separation of the tester Separation of the tester cDNA into two samples, cDNA into two samples, followed by twofollowed by two
♦ different adapter ligationdifferent adapter ligation
♦ Two successive Two successive subtractive hybridizationsubtractive hybridization
♦ PCR amplification of PCR amplification of target sequencestarget sequences
♦ Construction and Construction and screening of the subtracted screening of the subtracted library. library.
Advantages of SSH:
• Allows the detection of low abundance differentially expressed transcripts.
• Require one round of subtraction hybridization
•Requires as little as 2 micro g. of each genomic DNA/RNA sample.
• Well suited to high throughput sampling of all DNA/RNA that differs between two bacterial strains/conditions.
Disadvantages of SSH:
•There are a lot of false positive clones.
•Only a pair wise comparison between two population.
RNA
mRNA
Digested cDNA
For better understanding….,
Adaptor ligation efficiency
Primary and secondary PCR
Subtraction efficiency PCR
Colony PCR
Blue white colonies
Screening
Forward subtracted probe
Reverse subtracted probe
Thank you..,Thank you..,
Suppression PCR
DDPCR (Differential display PCR)
works by systematic amplification of the 3' terminal portions of mRNAs and resolution of those fragments on a DNA sequencing gel. Using anchored primers designed to bind to the 5' boundary of the poly-A tails for the reverse transcription, followed by PCR amplification with additional upstream primers of arbitrary sequences,
PAGE of ddPCR product
Immuno Screening
PHAGE DISPLAY SYSTEM: pEZM3
Biopanning of target clone in Phage display library
Biopanning involves 4 major steps for peptide selection:Making the display library; Capture; washing and elution
Application of Phage Display
1. Receptor / ligand interactions:use orphan receptor to screen for peptide ligands
Generation of DNA fragmentsRE/cDNA/Mechanical shear
Selection of vectorRestriction and ligation
Linkers - Linkers are short stretches of double stranded DNA of length 8-14 bp and have recognition site for 3-8 RE.
These linkers are ligated to blunt end DNA by ligase. Because of the high concentration of these small molecules present in the reaction, the ligation is very efficient when compared with blunt-end ligation of large molecules.
Adapters:- Adapters are linkers with cohesive ends or a linker digested with RE, before ligation.By adding adaptors to the ends of a DNA, sequences that are blunt can be converted into cohesive ends.