PLASMID RECOMBINANT TECHNIQUE 1. Isolate plasmid (vector) DNA and human DNA.

Prepare two kinds of DNA: bacterial plasmid as the vector and human DNA containin the ene of interest. !he plasmid is taken from E. coli and carr"in two ene: ampR: iven r#sistance to ampicillin

antibiotic in E. coli lacZ : codes $% alaktosidase& that breakdown lactose '. Insert human DNA into plasmid: a. (ut both DNAs with the same restriction en)"me. *estriction en)"me makes stick" ends at the both DNA. b. +i, the DNAs: the" -oin b" base pairin & also this one& -oin with the ene of interest. c. Add DNA li ase to bond covalentl". And then it makes DNA recombinant. .. Put plasmids into lacZ bacteria b" transformation. /. (lone cells: a. Plate cells onto medium with ampicillin and ,% al. Ampicillin: makes sure that onl" cell with DNA recombinant can row& because onl" this cell has ampR ene that resistance to ampicillin. 0% al: makes easier to identif" bacteria with DNA recombinant.

b. Identif" clones of cells containin recombinant plasmids b" their abilit" to row in presence of ampicillin and their white color 1. Identif" clone carr"in ene of interest.

GENE THERAPY 2ene therap" involves the introduction of enetic material into a cell to treat disease. +an" of the conditions treated in this wa" are enetic diseases that occur when enes malfunction. A common approach in ene therap" is to identif" a malfunctionin ene and suppl" the patient with specific enes on or off& functionin copies of that ene. 3ther approaches include switchin introducin enes to kill cancer cells& to suppress the blood suppl"& or to

tumours b" inhibitin

stimulate the immune s"stem to attack certain t"pes of cells. 4hichever approach is used& the aim of ene therap" is to introduce therapeutic material into the tar et cells& for this to become active inside the patient and e,ert the intended therapeutic effect. At present& ene therap" is still at the clinical research sta e. Gene therapy: so at!" an# $er %&!ne $ene therapy So at!" Ce&& Gene Therapy +an" enetic diseases ma" be able to be treated b" correctin the defective enes& b" ene therap". 2ene therap" is a therapeutic techni5ue in which a functionin ene is inserted into the cells of a patient to correct an inborn enetic error or to provide a new function to the cell. It means the enetic modification of DNA in the bod" cells of an individual patient& directed to alleviatin disease in that patient.

!here have been several hundred human ene therap" clinical trials in man" countries (includin 67A& 86& (anada& (hina& 9apan& New :ealand&etc)& involvin over ;<<< patients world%wide& for several different diseases includin several cancers. 7omatic cell ene therap" involves in-ection of =health" enes= into somatic (bod") cells of a patient. !he DNA chan e is not inherited to children. !he first human ene therap" protocol be an in 7eptember 1>>< that successfull" treated adenosine deaminase deficienc" (ADA) disease. ?rom 1>@> until 7eptember 1>>> there were thousands of patients in trials and no one died because of the e,periments. 1@ "ear%old 9esse 2elsin er died at the 6niversit" of Penns"lvania (67A) on 1A 7eptember 1>>>& four da"s after receivin a relativel" hi h dose of an e,perimental ene therap". Bis death was the result of a lar e immune reaction to the eneticall" en ineered adenovirus that researchers had infused into his liver. !here was much review of the procedures for safet" followin that case. 2ene therap" is still an e,perimental therap"& but if it is safe and effective& it ma" prove to be a better approach to therap" than man" current therapies& because ene therap" cures the cause of the disease rather than merel" treatin the s"mptoms of a disease. Also& man" diseases are still incurable b" other means& so the potential benefit is savin life. Ger %&!ne $ene therapy At the present ene therap" is not inheritable. 2erm cells are cells connected with and sperm cells and the reproduction& found in the testis (males) and ovar" (females)& i.e. 8

cells that ive rise to them. 2erm%line ene therap" tar ets the erm cells. !his t"pe of therap" ma" also mean in-ectin DNA to correct& modif" or add DNA into the pronucleus of a fertili)ed e . !he latter technolo " would re5uire that fertili)ation would occur in vitro usin the usual IC? procedures of super%ovulation and fertili)ation of a number of e embr"o=s chromosomes. HUMAN CLONING In order to stud" the structure of a ene it is necessar" to isolate it from other enes. !he most common method of achievin this is to make a ene librar" and screen it for clones containin the ene of interest. Dibraries can be made from the entire enome of the or anism ( enomic) or -ust from those enes that are e,pressed (transcribed) in a particular tissue at a cells prior to micromanipulation for DNA transfer and then embr"o transfer to a mother after checkin the

particular time (cDNA). DNA molecules ma" be manipulated b" usin restriction endonucleases. !hese en)"mes reco ni)e specific se5uences in the DNA molecule and cut it wherever the" occur. ?ra ments of DNA ma" be -oined to ether b" DNA li ase. ?ra ments of DNA ma" be li ated into either plasmids (autonomousl" replicatin circular DNA molecules found in bacteria and "east) or bacteriopha es (viruses that infect bacteria) and propa ated b" transformation of 8scherichia coli. !he forei n DNA is faithfull" replicated b" the host bacteria and individual clones ma" be selected b" colon" h"bridi)ation with a suitable probe. Plasmid or pha e DNA containin the fra ment of interest can be isolated from bacteria for se5uencin or further manipulation. (lonin is the process of ase,uall" producin a roup of cells (clones)& all eneticall" identical to the ori inal ancestor. !he word is also used in recombinant DNA procedures manipulation to produce

multiple copies of a sin le ene or se ment of DNA. It is more commonl" known as the production of a cell or an or anism from a somatic cell of an or anism with the same nuclear enomic ( enetic) characters % without fertili)ation. A clone is a collection of cells or or anisms that are eneticall" identical. 7ome ve etables are made this wa"& like aspara us& or flowers like orchids. Buman repro#'"t!(e "&on!n$ is the production of a human fetus from a sin le cell b" ase,ual reproduction. In '<<1 a cloned embr"o was reported made b" nuclear transfer& thou h in 1>>. cloned embr"os were made b" splittin human embr"os. In the late 1>><s reproductive clonin was used to produce clones of the adults of a number of mammalian species& includin sheep& mice and pi s. !he most famous of these was Doll"& the sheep. +an" countries rushed to outlaw the possibilit" of reproductive clonin in humans. +ost mammalian embr"os can onl" be split into '%/ clones& after that the cells lack the abilit" to start development into a human bein .

Therape't!" "&on!n$ is the clonin of embr"os containin DNA from an individual=s own cell to enerate a source of embr"onic stem (87) cell%pro enitor cells that can differentiate into the different cell t"pes of the bod". 87 cells are capable of eneratin all cell t"pes& unlike multipotent adult%derived stem cells which enerate man" but not all cell t"pes. !he aim is to produce health" replacement tissue that would be readil" available. 7ince it is from the same bod" it is immunocompatible so that the recipients would not have to take immunosuppressant dru s for the rest of their lives& as the" do if the" receive an or an from another person.

AD)ANTAGES O* BIOTECHNOLOGY Phar a"o$eno !"s Pharmaco enomics is the stud" of how an individual=s enetic inheritance affects the bod"=s response to dru s. !he term comes from the words pharmacolo " and enomics and is thus the intersection of pharmaceuticals and enetics. Pharmaco enomics holds the promise that dru s mi ht one da" be tailor%made for individuals and adapted to each person=s own enetic makeup. 8nvironment& diet& a e& lifest"le& and state of health all can influence a person=s response to medicines& but understandin an individual=s enetic makeup is thou ht to be the ke" to creatin personali)ed dru s with reater efficac" and safet". Pharmaco enomics combines traditional pharmaceutical sciences such as biochemistr" with annotated knowled e of enes& proteins& and sin le nucleotide pol"morphisms. 3ne can anticipate the benefits of Pharmaco enomics& which are as follows: E More Po+er,'& Me#!"!nes Pharmaceutical companies will be able to create dru s based on the proteins& en)"mes& and *NA molecules associated with enes and diseases. !his will facilitate dru discover" and

allow dru makers to produce a therap" more tar eted to specific diseases. !his accurac" not onl" will ma,imi)e therapeutic effects but also decrease dama e to nearb" health" cells. E Better- Sa,er Dr'$s the *!rst T! e Instead of the standard trial%and%error method of matchin patients with the ri ht dru s& doctors will be able to anal")e a patient=s enetic profile and prescribe the best available dru therap" from the be innin . Not onl" will this take the uesswork out of findin the ri ht dru & it will speed recover" time and increase safet" as the likelihood of adverse reactions is eliminated. Pharmaco enomics has the potential to dramaticall" reduce the estimated 1<<&<<< deaths and ' million hospitali)ations that occur each "ear in the 6nited 7tates as the result of adverse dru response (1). E More A""'rate Metho#s o, Deter !n!n$ Appropr!ate Dr'$ Dosa$es (urrent methods of basin dosa es on wei ht and a e will be replaced with dosa es based on a person=s enetics %%how well the bod" processes the medicine and the time it takes to metaboli)e it. !his will ma,imi)e the therap"=s value and decrease the likelihood of overdose. E A#(an"e# S"reen!n$ ,or D!sease Fnowin one=s enetic code will allow a person to make ade5uate lifest"le and environmental chan es at an earl" a e so as to avoid or lessen the severit" of a enetic disease. Dikewise& advance knowled e of particular disease susceptibilit" will allow careful monitorin & and treatments can be introduced at the most appropriate sta e to ma,imi)e their therap". E Better )a""!nes Caccines made of enetic material& either DNA or *NAG promise all the benefits of e,istin vaccines without all the risks. !he" will activate the immune s"stem but will be unable to cause infections. !he" will be ine,pensive& stable& eas" to store& and capable of bein en ineered to carr" several strains of a patho en at once.