ON

ENZYMES USED IN GENETIC ENGINEERING

SUBJECT:- CONCEPTS IN BIOTECHNOLOGY SUBJECT CODE:-BTC-012

SUBMITTED TO:Mr. HARSH KUMAR (Lect. In BIOTECHNOLOGY)

SUBMITTED BY:ASHUTOSH B.Tech(Bio Tech)- 3rd sem Section- B ROLL NO. R109A30 Reg. NO.1040070139

LOVELY PROFESSIONAL UNIVERSITY

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PREFACE

Genetic engineering is a new technology that combines genes from totally unrelated species, in combinations not possible using conventional breeding methods. Genes from an animal, say, a fish, can be put into a plant, a strawberry for instance.. So different type of enzymes are used in genetic engineering In fact this is an actual example of an attempt to "improve" strawberry plants. The fish gene is supposed to make the strawberries more resistant to frost by causing the strawberry plant to produce a form of antifreeze which the fish normally produces to endure cold ocean conditions. The primary goal of this term parer has been to provide useful information about genetic engineering in a simple and illustrative language . The organization of the material Is largely traditional and the order in which the content arranged, is a matter of personal choice. I hope you will enjoy reading.

 Thank you

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ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the possibility to complete this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY PROFESSIONAL UNIVERSITY for giving me permission to commence this Term paper, to do the necessary research work and to use departmental data. I have furthermore to thank to Mr. HARSH KUMAR , Lect. In CONCEPTS OF BIOTECHNOLOGY who gave and confirmed this permission and encouraged me to go ahead with my term paper. I am deeply indebted to my supervisor Lect. HARSH SIR whose help, stimulating suggestions and encouragement me in all the time of research for and writing of this term paper.

Thank you

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CONTENTS
 1.INTRODUCTION  2.INTRODUCTION TO GENETIC ENGINEERING  3. ENZYMES IN GENETIC ENGINEERING  4.RESTRICTION ENZYME AND THEIR MODE OF ACTION  5.DNA LIGASE  6.PROPERTIES OF RESTRICTION ENZYME  7.EXAMPLE OF RESTRICTION ENZYME

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INTRODUCTION

Biotechnology has to do with the manipulation of organisms to get useful products. One of the basis of biotechnology is genetic transformation. Genetic transformation occurs when DNA is taken in and expressed by a cell from a living organism. Hence different type of enzymes are required for the same,like restriction enzyme, ligase. In this term paper we will discuss about the different enzymes used in genetic engineering, their mode of action along with their structure.Different type of enzymes work in different manner to the DNA of host.

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Genetic Engineering

Who would have thought that a tomato could possess characteristics of a fish? What about a plant possessing characteristics of a firefly; or a pig with human traits? These things may sound like science experiments gone wrong, but in truth, these are products of experiments that went well. The fish-like tomato and others are results of genetic modification or genetic manipulation, which are more commonly known as genetic engineering. Genetic engineering is the process of taking genes and segments of DNA from one species and putting them into another species, thus breaking the species barrier and artificially modifying the DNA of various species. These changes in DNA result in an alteration of reproductive and hereditary processes of the organisms since the process is irreversible and the organism's offspring will also possess this unique DNA (Levine).

Enzymes
Enzymes used in genetic engineering such as restriction endonuclease (biological scissors), DNA polymerase (for replication of DNA), reverse transcriptase (to make DNA from RNA) and ligase (to ligate two DNA fragments) are produced by recombinant DNA technology (by cloning in high copy number plasmids in bacteria). Other enzymes now routinely produced by recombinant DNA technology are rennin (an enzyme used in cheese making mentioned above), lipase (cheese making),  amylase (beer making), bromelain (meat tenderizer, juice clarification), catalase (antioxidant in food), cellulase (alcohol and glucose production), and protease (detergents).

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Enzymes and Genetic Engineering

1. In genetic engineering, scientists use restriction enzymes to isolate a segment of DNA that contains a gene of interest, for example, the gene regulating insulin production. 2. A plasmid extracted from its bacteria and treated with the same restriction enzyme can hybridize with this fragment’s “sticky” ends of complementary DNA. 3. The hybrid plasmid is reincorporated into the bacterial cell, where it replicates as part of the cell’s DNA. 4. A large number of daughter cells can be cultured and their gene products extracted for human use.

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Restriction Enzymes

The first major breakthrough on the road to genetic engineering came with work done on restriction endonucleases by Herbert Boyer of the University of California at San Francisco. As defined by Karl Drlica in Understanding DNA and Gene Cloning: A Guide for the Curious, restriction endonucleases "are a group of enzymes [a special type of protein] that . . . occur naturally in a large number of different bacterial species, serving as part of the natural defense mechanism that protects bacterial cells against invasion by foreign DNA molecules such as those contained in viruses."15 When, for example, a virus attacks a single-celled bacterium, restriction endonucleases are unleashed and go to work, cutting the invading DNA into small, nonthreatening pieces. "Crucial to this protective device is the ability of the nuclease to discriminate between its own DNA and the invading DNA; otherwise the cell would destroy its own DNA," Drlica says. This recognition process involves two elements. First there are specific nucleotide sequences [As and Ts, Cs and Gs] that act as targets for the nuclease. These are called the restriction sites. Second, there is a protective chemical signal that can be placed by the cell on all the target sequences that happen to occur in its own DNA. The signal modifies the DNA and prevents the nuclease from cutting. Invading DNAs, lacking the protective signal, would be chopped by the nuclease.16 Thus, restriction enzymes have the remarkable ability to recognize specific arrangements of DNA base pairs—As and Ts, Gs and Cs. They also have the capacity to act like a molecular scalpel, severing the DNA at exactly the spot where they detect this sequence of genetic letters. Restriction enzymes are a powerful tool because there are thousands of them, and each one acts only on a unique arrangement of As and Ts and Cs and Gs.

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The science of genetic engineering originated in the late 1960s and early 1970s with the discovery of restriction enzymes (Avise, 1998). While investigating how viruses and rings of deoxyribonucleic acid (DNA) called plasmids infect bacterial cells, recombine, and reproduce themselves, scientists discovered that bacteria make enzymes, called restriction enzymes, that cut DNA chains at specific sites. Restriction enzymes recognize particular stretches of nucleotides arranged in a specific order and cut the DNA in those regions only. Each restriction enzyme recognizes a different nucleotide sequence. Thus, restriction enzymes form a molecular tool kit that allows the chromosome to be cut into various desired lengths, depending on how many different restriction enzymes are used. Each time a particular restriction enzyme or set of restriction enzymes is used, the DNA is cut into identical pieces of the same number allowing for precise replication.

Restriction enzymes make it possible to remove a bit of DNA from one organism's chromosome and to insert it into another organism's chromosome This allows for the production of new combinations of genes that may not exist in nature. For example, a

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human gene can be inserted into a bacterium or a bacterial gene into a plant. So far, however, there are limits to this ability. Jurassic Park fantasies notwithstanding, scientists are currently unable to create a whole new organism starting solely with a test tube full of nucleotides. They must start with the complete genetic material of an already existing organism. Thus, genetic engineering allows the addition of only one or a small number of new characteristics to an organism that remains essentially the same. In addition, only characteristics that are determined by one or a few genes can be transferred. The current knowledge of behavioral genetics is not sufficiently advanced to enable scientists to transfer behavioral traits, such as intelligence, that are a complex mixture of many genes and ontogenetic factors.

Mode of Action of Restriction Enzymes –
The restriction enzyme binds to the recognition site and checks for the methylation (presence does of methyl group on the not DNA at a specific nucleotide). cut. If there is methylation in the recognition sequence, then, it just falls off the DNA and If only one strand in the DNA molecule is methylated in the recognition sequence and the other strand is not methylated, then RE (only type I and type III) will methylate the other strand at the required position. The methyl group is taken by the RE from S-adenosyl methionine by using modification site present in the restriction enzymes. However, type II restriction enzymes take the help of another enzyme called methylase, and methylate the DNA. Then RE clears the DNA. If there is no methylation on both the strands of DNA, then RE cleaves the DNA. It is only by this methylation mechanism that, RE, although present in bacteria, does not

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cleave the bacterial DNA but cleaves the foreign DNA. But there are some restriction enzymes which function exactly in reverse mode. They cut the DNA if it is a methylate These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends. The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at specific base sequences, 4-8 base pairs long, called recognition sequences. Restriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict” viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments.

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DNA Ligase This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments. The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete.

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DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different

Properties of restriction enzymes Ends
As illustrated, restriction enzymes invariably cut DNA in such a way as to leave a 3' hydroxyl on one end, and a 5' phosphate on the other. In addition, most (but not all) enzymes recognize a symmetrical site (see the figures below). Another interesting property of restriction enzymes is that while they often recognize a symmetrical site, they do not always cut at the axis of symmetry. For instance, the enzyme EcoRI (from Escherichia coli strain RY13 (I guess they didn't want to put all those letters in the name of the enzyme) and pronounced "echo are one"), recognizes the site GAATTC and cuts the DNA between the G's and the A's in a manner depicted below. Note that the cut produces an overhanging 5' single-stranded end of four nucleotides on each of the two pieces that are newly liberated (this is clearer if you look at the next two illustrations). Similarly, the enzyme BglII (from the microorganisms, Bacillus globiggi and universally and irreverently pronounced BAGEL TWO) recognizes the sequence AGATCT and cuts between the first A and G residues.Notice that BglII also yields a single-stranded 5' end. A third enzyme, BamHI (from the bacterium Bacillus amyloliquifaciens) cuts similarly. In fact, the four nucleotide single-stranded ends are the same for both BglII and BamHI. Moreover, there are at least two other six cutting enzymes that have been discovered that leave the same four nucleotide overhang: BclI and XhoII. These overhanging ends are 13

very useful because -- under the proper conditions -- they may base pair with each other. In fact, because of their affinity for one another, they are often called cohesive or sticky ends. Moreover, if molecules with these ends are treated with the appropriate enzyme -DNA ligase -- their phosphodiester bonds may be rejoined (ligated). When two ends that originate from digestion by a single enzyme are ligated, the resulting molecule can be cut by the same enzyme again. But if the ends of a DNA molecule that originated with a BamHI cut and a BglII cut are joined together, the new sequence will not be cut with either enzyme (I'll ask you to explain why this is so in class). Note that all restriction endonucleases do not generate 5' single-strand overhangs. In fact, some don't even produce an overhang at all. Several enzymes -- like SacI -- produce 3' single-stranded sticky ends. And some enzymes -- like PvuII -- cut at the axis of symmetry, leaving perfectly aligned ends. DNA molecules without overhangs are said to have blunt ends. In addition there are restriction enzymes that cleave DNA some distance away from the sequence that they recognize. For example the enzyme HgaI makes staggered cuts that lie 5 and 10 nucleotides away from a 5 base pair sequence, GACGC. This leaves 5' overhanging ends, but, in contrast to the enzymes described above, these will be different almost every time the enzyme cuts .

Examples of restriction enzymes include:
Enzyme Source EcoRI EcoRII Escherichia coli Escherichia coli Recognition Sequence 5'GAATTC 3'CTTAAG 5'CCWGG 3'GGWCC Cut 5'---G AATTC---3'

3'---CTTAA G---5' 5'--- CCWGG---3' 3'---GGWCC ---5'

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BamHI HindIII TaqI

Bacillus amyloliquefaciens Haemophilus influenzae Thermus aquaticus

5'GGATCC 3'CCTAGG 5'AAGCTT 3'TTCGAA 5'TCGA 3'AGCT 5'GCGGCCGC 3'CGCCGGCG 5'GANTC 3'CTNAG 5'GATC 3'CTAG 5'CAGCTG 3'GTCGAC 5'CCCGGG 3'GGGCCC 5'GGCC 3'CCGG 5'AGCT 3'TCGA 5'GATATC 3'CTATAG 5'GGTACC 3'CCATGG 5'CTGCAG 3'GACGTC 5'GAGCTC 3'CTCGAG 5'GTCGAC 3'CAGCTG 5'AGTACT 3'TCATGA 5'GCATGC

5'---G

GATCC---3'

3'---CCTAG G---5' 5'---A AGCTT---3' 3'---TTCGA A---5' 5'---T CGA---3' 3'---AGC T---5' 5'---GC GGCCGC--3' 3'---CGCCGG CG--5' 5'---G ANTC---3' 3'---CTNA G---5' 5'--- GATC---3' 3'---CTAG ---5' 5'---CAG CTG---3' 3'---GTC GAC---5' 5'---CCC GGG---3' 3'---GGG CCC---5' 5'---GG CC---3' 3'---CC GG---5' 5'---AG CT---3' 3'---TC GA---5' 5'---GAT ATC---3' 3'---CTA TAG---5' 5'---GGTAC C---3' 3'---C CATGG---5' 5'---CTGCA G---3' 3'---G ACGTC---5' 5'---GAGCT C---3' 3'---C TCGAG---5' 5'---G TCGAC---3' 3'---CAGCT G---5' 5'---AGT ACT---3' 3'---TCA TGA---5' 5'---G CATGC---3'

NotI

Nocardia otitidis

HinfI Sau3A PovII* SmaI* HaeIII* AluI*

Haemophilus influenzae Staphylococcus aureus Proteus vulgaris Serratia marcescens Haemophilus aegyptius Arthrobacter luteus

EcoRV* Escherichia coli KpnI[30] PstI[30] SacI[30] SalI[30] ScaI[30] SphI
[30]

Klebsiella pneumoniae Providencia stuartii Streptomyces achromogenes Streptomyces albus Streptomyces caespitosus Streptomyces

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phaeochromogenes StuI [31][32] Streptomyces tubercidicus XbaI[30] Xanthomonas badrii

3'CGTACG 5'AGGCCT 3'TCCGGA 5'TCTAGA 3'AGATCT

3'---CGTAC G---5' 5'---AGG CCT---3' 3'---TCC GGA---5' 5'---T CTAGA---3' 3'---AGATC T---5'

* = blunt ends N = C or G or T or A W = A or T

The

utility

of

the

restriction

enzymes

The discovery of these many restriction endonucleases have allowed genetic engineers to cut pieces of DNA at specific sites and into defined sizes. The result has been that a scientist can work with a collection of molecules all of the same size and with ends of known sequence. Restriction enzymes have proved to be valuable analytical and diagnostic tools as well.

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References
• TEXT BOOKS: 1. Biotechnology-expanding horizons: b.d.singh. Kalyani publishers . 2nd edition  2. Molecular biotechnology: s.b.parimrose. Panima publication. 2nd edition

• INTERNET: Www. Google.com  www.bionewsonline.com  www.library.thinkquest.org  Www.yahoo.com  www.biotech.about.com/od/whatisbiotechnology  www.geneticengineering.org

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