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

INTRODUCTION OF RECOMBINANT DNA INTO THE CELL

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

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

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

obtaining low abundance obtaining low abundance genes;genes;

3. High specificity3. High specificity

1. Generally, only 1. Generally, only two samples can two samples can be compared in be compared in one SSH;one SSH;

2. The results too 2. The results too depend on the depend on the efficacy of ligating efficacy of ligating adaptorsadaptors

MicroaMicroarrayrray

1. Smallest quantities of 1. Smallest quantities of samples; samples;

2. Highest efficacy; 3. 2. Highest efficacy; 3. High specificity; High specificity;

4. More than two samples 4. More than two samples can be analyzed in one can be analyzed in one experimentexperiment

1. In general, it 1. In general, it require some require some sequence sequence information in information in advance;advance;

2. Low abundance 2. Low abundance genes are difficult genes are difficult to detect; 3. High to detect; 3. High costcost

RDARDA 1. High specificity and 1. High specificity and low percentage of false low percentage of false positive; positive;

2. cDNA RDA can be 2. cDNA RDA can be used in mRNA without used in mRNA without polyApolyA

1. Low percentage of 1. Low percentage of obtaining the low obtaining the low abundance genes;abundance genes;

2. Repeated 2. Repeated procedures of procedures of enzyme cut and enzyme cut and ligation.ligation.

DD DD PCPCRR

1. Relatively smaller 1. Relatively smaller quantities of samples;quantities of samples;

2. High efficacy, and 2. High efficacy, and normally low normally low abundance genes can abundance genes can also be obtained; also be obtained;

3. More than two samples 3. More than two samples can be analyzed in one can be analyzed in one experimentexperiment

1. High percentage of 1. High percentage of false positive;false positive;

2. The amplified 2. The amplified products areproducts are

usually in the 3’-usually in the 3’-terminal regionterminal region

SHSH 1. Low cost; 1. Low cost; 2. Relatively longer genes 2. Relatively longer genes

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

2. Enzyme / substrate interactions:screen peptide inhibitors

3. DNA protein interaction

4. Identification of antigenic peptide

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.