SOURCE ORGANISM

GENE CLONING

ISOLATION OF PURIFIED DNA

?GENERATION OF FRAGMENTS WITH THE GENE OF INTEREST

Plasmid based vectors SELECTION OF VECTOR Phage based vectors Yeast expression vectors
PREPARATION OF VECTOR AND INSERT WITH COMPATIBLE ENDS

DNA MODIFYING ENZYMES

NUCLEASES, PHOSPHATASES, KINASES ETC.

LIGATION AND RECOMBINANT MOLECULE

INTRODUCTION OF RECOMBINANT DNA INTO THE CELL

SCREENING AND SELECTION OF THE DESIRED RECOMBINANT

INTRODUCTION OF RECOMBINANT DNA INTO THE CELL Transformation Transfection SCREENING AND SELECTION OF THE DESIRED RECOMBINANT Selection for recombinants

Selection 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 ln(1-P) N= ------------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

cDNA library

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 halflife is longer. These probes can be prepared in advance in bulk and stored at - 20C 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
Cloning of genes based on defferential expression patterns
DDPCR SSH (suppression subtractive hybridization)

Chromosomal location Map-based cloning (using genetic approach) Wait till you are taught Molecular markers

Complementary Genetics

1. Protein sequence is related to gene sequence

NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COOATG 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 Forward primer: 5-ATGGAT/CGCN-3 Reverse primer: 5-T/CTTNC/GT/ACCA-3 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

Complementary Genetics (cont.)

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

c. two copies of the template DNA made (72C)

3 5 5 3

Methods for discovering differentially expressed genes

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)

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.

Advantage and disadvantage of gene expression profile

Techni ques SSH Advantage 1. Relatively smaller quantities of samples; 2. High efficacy, especially efficient for obtaining low abundance genes; 3. High specificity Disadvantage 1. Generally, only two samples can be compared in one SSH; 2. The results too depend on the efficacy of ligating adaptors

Microa 1. Smallest quantities of 1. In general, it rray samples; require some 2. Highest efficacy; 3. sequence High specificity; information in 4. More than two samples advance; can be analyzed in one 2. Low abundance experiment genes are difficult to detect; 3. High cost RDA 1. High specificity and 1. Low percentage of low percentage of false obtaining the low positive; abundance genes; 2. cDNA RDA can be used 2. Repeated in mRNA without procedures of

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 cDNAs Digestion by a four base cutting restrictive enzyme Separation of the tester cDNA into two samples, followed by two different adapter ligation Two successive subtractive hybridization PCR amplification of target sequences Construction and screening of the subtracted 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.

For better understanding.,

RNA

mRNA

Digested cDNA

Adaptor ligation efficiency

Primary and secondary PCR

Subtraction efficiency PCR

Blue white colonies

Colony PCR

Screening

Forward subtracted probe

Reverse subtracted probe

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 2. Enzyme / substrate interactions: screen peptide inhibitors 3. DNA protein interaction 4. Identification of antigenic peptide

Generation of DNA fragments

RE/cDNA/Mechanical shear

Selection of vector Restriction 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.