Seminar on :- Constructing Contigs Sequencing VIDYA PRATISHTHAN'S VSBT COLLEGE OF AGRICULTURAL BIOTECHNOLOGY ( Affiliated to Mahatma Phule Krishi Vidyapeeth, Rahuri ) Vidyanagari , Baramati, Dist- Pune 413133 Seminar on :- Constructing Contigs Sequencing Presented by : Swapnil Gorakh Waghmare

Contents : Introduction Constructing contig Chromosome jumping Chromosome walking Applications

Introduction : A contig (from contiguous) is a set of overlapping DNA segments that together represent a consensus region of DNA . Contigs can thus refer both to overlapping DNA sequence and to overlapping physical segments (fragments) contained in clones depending on the context. The general approach in creation of contigs is to identify clones that have adjacent DNA segments for the CHROMOSOME; the members of contig must contain some overlapping regions to allow the precise determination of their location in the contig.

A contig represents a physical map of a chromosome. The cloned DNA fragment of a contig can be correlated with locations along a chromosome obtained from linkage or cytogenetic mapping. This would permit the alignment of the other members of the contig along the chromosome. YAC cloning vectors are used to make contigs of eukaryotic chromosomes. Therefore, YAC cloning is usually the first step in creating a physical map of genome.

Chromosome A B C d E F G H DNA FRAGMENTS PRODUCED FROM THE CHROMOSOME INDIVIDUAL PIECES CLONED INTO VECTORS E B H A F G G B C C d F 6 d 5 4 3 Vector 1 2 Fig.1: A collection of clones containing overlapping DNA inserts

Constructing contigs : Different Experimental Strategies can be employed to align the members of a contig. The strategies using chromosome jumping and Chromosome walking are briefly described below.

Chromosome Jumping : Chromosome jumping is used for rather larger DNA Fragment, usually of several hundred kb in size . Each clone in a jumping library contains the DNA sequences on one side each e.g., sequences 2 & 3, of two neighboring cutting sites of enzyme NotI, while a clone in linking library has the DNA sequence located on either side of a single NotI, site e.g., regions 1 and 2 in clone 1 and regions 3 and 4 in clone 2.

PROCEDURE FOR CONSTRUCTING OF A NotI JUMPING LIBRARY :- Genomic DNA is completely digested with NotI, and the resulting fragments are circularized in the presence of SupF marker. The circularized fragments are digested with a frequent cutting enzyme like BamHI to delete bulk of the DNA leaving only a small DNA sequence on either side of the SupF marker.

The resulting fragments are ligated in suitable λ vector and cloned. SupF is a tRNA gene that has a mutated anticodon, which recognizes a polypeptide chain termination codon generated by a supressor-sensitive mutation within a gene.

PROCEDURE FOR CONSTRUCTING OF A LINKING LIBRARY :- The genomic DNA is partially digested with a frequent cutting enzyme like Sau 3A and the fragment are circularized in the presence of supF marker. The circularized fragments are cut open with the enzyme NotI, and the linear fragments are integrated in a suitable λ vector.

Only those circles that have a NotI site will become linear and , hence , integrate in the λ vector. It is important to note that the regions surrounding a single NotI site are present in one clone.

S S S S S N N GENOMIC DNA 1 2 3 4 Not I COMPLETE DIGETION PARTIAL DIGETION WITH Sau 3A N 1 2 N 3 4 CIRCULARIZATION IN PRESENCE OF SupF MARKER 2 1 B B B B 3 2 3 4

JUMPING LIBRARY LINKING LIBRARY Fig .2 Jumping and linking library 1 2 DIGEST WITH NotI DIGEST WITH BamHI 1 2 2 3 3 4 VECTOR LINEARIZED WITH BamHI VECTOR LINEARIZED WITH NotI LIGATE INTO λ VECTOR 1 2 (CLONE 1) 2 3 (CLONE 2) VECTOR 3 4 JUMPING LIBRARY LINKING LIBRARY Fig .2 Jumping and linking library

Each clone of a linking library hybridizes with two different clones of the jumping library and vice-versa. But each linking library clone will hybridize with only one YAC clone, while each clone of jumping library will hybridize with two different YAC clones. Each clone of linking and jumping libraries is hybridized separately with all the YAC clones obtained from the target chromosome.

GENOMIC DNA FRAGMENTS BY Not I The data obtained from these hybridizations are pooled together to determine the correct order of various YAC clones leading to the creation of a contig for the chromosome. JUMPING CLONES 2 3 GENOMIC DNA FRAGMENTS BY Not I 1 2 3 4 LINKING CLONES 3 4 1 2 Fig. 3 Determination of correct order of DNA fragments

Chromosome Walking : Generally, a cosmid library is used for chromosome walking; each clone in such a library may be expected to have a DNA insert of , on an average , 40 kb. In chromosome walking, one begins with a DNA fragment that contains a known gene / genetic marker, e.g., an RFLP marker.

The sequence located at one end of this fragment is used to identify a clone that has such a DNA insert, which partly overlaps the first fragment. Now the other (nonoverlapping) end of this new DNA fragment is used as probe. In this way, one continues to move step-by-step toward another end of chromosome.

Procedure of Chromosome walking : Isolation of a DNA fragment (fragment 1) containing a known gene or marker . This fragment provides the starting point for the chromosome walk. A restriction map of this fragment is prepared. A small segment representing one end of this original fragment (fragment 1) is isolated And cloned , this is called subcloning.

This subclone is now used as a probe for the identification of such clone in the genomic library that overlap fragment 1. The clone identified in this way will contain such a DNA insert that overlaps fragment-1 preferably at one end; the new genomic fragment may be referred to as fragment -2.

A restriction map of fragment 2 is prepared, and the sequence at the other end of this fragment is now used as a probe to identify clones having DNA insert overlapping with fragment 2. The DNA fragment obtained from such clones will be overlapping with fragment 2 preferably at one end ; this new genomic fragment may be called as fragment 3. the process of step 3 is repeated till we reach one end of the chromosome.

{ { A DNA SEGMENT FROM A CHROMOSOME Fragment 1 PREPARE RESTRICTION MAP USE THIS SEQUENCE AS PROBE { PROBE (PRODUCED BY SUBCLONING) HYBRIDIZE WITH CLONES FROM GENOMIC LIBRARY Fragment 2 ISOLATE FRAGMENT 2 PREPARE RESTRICTION MAP OF FRAGMENT 2 PROBE { USE THIS SEQUENCE AS PROBE

Fig 4. Chromosome Walking PROBE (PRODUCED BY SUBCLONING) HYBRIDIZE WITH CLONES FROM GENOMIC LIBRARY Fragment 3 ISOLATE FRAGMENT 3 PREPARE RESTRICTION MAP OF FRAGMENT 3 PROBE CONTINUE Fragment 1 Fragment 2 Fragment 3 SUCCESSIVE FRAGMENTS Fig 4. Chromosome Walking

APPLICATIONS : For the construction of physical map. For the preparation of overlapping clones. For the determination of specific region on the chromosome. For the sequencing of genome which is not in order.

Reference : B.D. Singh BIOTECHNOLOGY EXPANDING HORIZONS (fourth edition), Kalyani Publishers, pp 139-142, 904-905.

Acknowledgement I would like to express my special thanks of gratitude to my teacher Mrs. Priya Kakade as well as our principal Dr. A.S. Patil gave me the golden opportunity to do this wonderful seminar on the topic CONSTRUCTING CONTIGS SEQUENCING.

THANK YOU