Molecular maker aided breeding

Molecular maker aided breeding : 

Molecular maker aided breeding Presented by Birendra kumar IInd yr MSc BT

content : 

content Introduction Need of molecular marker RFLP map RAPD marker SCAR ESTs Conclusion

Introduction : 

Introduction Production of transgenic plant are of great importance. Involves the insertion of gene of interest(GI) for its translation. GI may be from different species or genera Insertion may be through physical, chemical or biological method. Isolation of these gene required several steps and need to be check

Terminology : 

Terminology Restriction site Restriction enzyme : polymerase, ligase, endonuclease PCR Molecular Markers Blotting

Molecular makers : 

Molecular makers They are specific genes which is used to check the efficiency and transformation. They may be antibiotic resistance like pen*, amp* etc or enzymes encoding gene (ENG) ENG are preferred for plant and animal transformation. Most widely used gal*

Dna markers : 

Dna markers RFLP : Restriction fragment length polymorphisms RAPD: Randomly Amplified Polymorphic DNA ESTs: Express Sequence Tags SCARS: Sequence Characterized Amplified Region SSR: Simple Sequence Repeat ALFP: Amplified Length Fragment Polymorphic DNA STS: Sequence Tag Site SNP: Single Nucleotide Polymorphisms CAPS: Cleaved Amplified Polymorphism Sequence

RFLP –Restriction Fragment Length Polymorphism : 

RFLP –Restriction Fragment Length Polymorphism restriction analysis of DNA by its digestion with restriction endonucleases (RE) in specific restriction sites in the case the sequence difference (polymorphism) creates or disturbs a specific site for RE, after restriction, fragments with different sizes are formed. RE recognize variously short nucleotides sequences (4,6,8), in which then they digest covalent phosphodiester bonds It helps in pinpointing the exact location and determining the identity or relatedness of individuals.

Slide 10: 

Restriction Fragment Length Polymorphism (RFLP) Analysis “DNA Fingerprinting”used in modern forensics Suspect Evidence Victim

RFLP maps : 

RFLP maps

RAPD marker : 

RAPD marker Randomly Amplified Polymorphic DNA a method based on PCR developed in 1990. RAPD is different from conventional PCR as it needs one primer for amplification. The size of primer is normally short (10 nucleotides), and therefore, less specific. the primers can be designed without the experimenter having any genetic information for the organism being tested. Genomic DNA normally has complimentary sequences to RAPD primers at many locations. If two of these locations are close to each other (<3000bp), and the sequences are in opposite orientation, the amplification will be established. This amplified region is said as a RAPD locus. Normally, a few (3-20) loci can be amplified by one single RAPD primer.

Slide 14: 

RAPD marker is a dominant marker. Presence of a DNA band is dominant; absence of a DNA band is recessive. DNA bands of different sizes are assumed to be amplified products from different RAPD loci. HOMOLOGY TEST FOR FRAGMENTS OF SIMILAR MOBILITY IN RAPD PROFILES

Modification of rapd : 

Modification of rapd AP-PCR- (Arbitrary Primed PCR). similar to RAPD, involves two cycles of low-stringency amplification, followed by cycles conducted at higher stringency, using primer of arbitrary sequence. DAMD- (Directed Amplification of Minisatellite Region DNA) technique for detecting polymorphisms using VNTR core sequences as primers for PCR ISSR (Inter-Simple Sequence Repeat). A PCR-based molecular marker assay of genomic sequence lying between adjacent microsatellites (SSRs). Primers carrying, at their 3'-end, sequence complementary to the repeat unit of the microsatellite will amplify this genomic DNA.

Sequence Characterized Amplified Region (SCAR) : 

Sequence Characterized Amplified Region (SCAR) SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. Obtaining a codominant marker may be an additional advantage of converting RAPDs into SCARs.

Expressed Sequence Tag(ESTs) : 

Expressed Sequence Tag(ESTs) a tiny portion of an entire gene that can be used to help identify unknown genes and to map their positions within a genome. ESTs provide researchers with a quick and inexpensive route for discovering new genes, for obtaining data on gene expression and regulation, and for constructing genome maps. Today, researchers using ESTs to study the human genome find themselves riding the crest of a wave of scientific discovery the likes of which has never been seen before.

What Are ESTs and How Are They Made? : 

What Are ESTs and How Are They Made? EST is a short sub-sequence of a transcribed cDNA sequence.They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination.The identification of ESTs has proceeded rapidly, with approximately 65,9 million ESTs now available in public databases (e.g. GenBank 18/6/2010, all species). An EST is produced by one-shot sequencing of a cloned mRNA (i.e. sequencing several hundred base pairs from an end of a cDNA clone taken from a cDNA library). The resulting sequence is a relatively low quality fragment whose length is limited by current technology to approximately 500 to 800 nucleotides. Because these clones consist of DNA that is complementary to mRNA, the ESTs represent portions of expressed genes.

What Are ESTs and How Are They Made? : 

What Are ESTs and How Are They Made? They may be present in the database as either cDNA/mRNA sequence or as the reverse complement of the mRNA, the template strand. ESTs can be mapped to specific chromosome locations using physical mapping techniques, such as radiation hybrid mapping, Happy mapping, or FISH. Alternatively, if the genome of the organism that originated the EST has been sequenced one can align the EST sequence to that genome using a computer.