Biotechnology
9.1 Principles of Biotechnology 
9.2 Tools of Recombinant DNA Technology
9.3 Processes of Recombinant DNA Technology
Introduction
Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.”
Explanation :
hum living organisms (jaise bacteria, plants, animals) ya unke enzymes ka use karte hain
taki useful products (jaise medicine, food) ya processes bana sakein
Simple: Living cheezon ka use karke human ke kaam ki cheez banana = Biotechnology
“In this sense, making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology.”
Explanation :
curd (dahi), bread, wine banana bhi biotechnology hai
kyunki ye sab microorganisms (bacteria/yeast) ki help se bante hain
Example:
Dahi → bacteria se
Bread → yeast se
“However, it is used in a restricted sense today, to refer to such of those processes which use genetically modified organisms to achieve the same on a larger scale.”
Explanation :
Lekin aaj kal biotechnology ka meaning thoda narrow (restricted) ho gaya hai:
Ab biotechnology ka matlab hota hai:
Genetically Modified Organisms (GMO) ka use
aur kaam large scale (industry level) par karna
Normal dahi banana = old biotech
Gene change karke insulin banana = modern biotech
“Further, many other processes/techniques are also included under biotechnology.”
Explanation :
Biotechnology me sirf GMO hi nahi, aur bhi bahut techniques aati hain
Matlab: field kaafi wide hai
“For example, in vitro fertilisation leading to a ‘test-tube’ baby…”
Example 1:
In vitro fertilisation (IVF) = lab me fertilization karna
isse test tube baby hota hai
Matlab: body ke bahar fertilization
“synthesising a gene and using it…”
Example 2:
Lab me gene banana (synthetic gene)
phir usko use karna
“developing a DNA vaccine…”
Example 3:
DNA vaccine banana
jo body ko disease se bachata hai
“or correcting a defective gene, are all part of biotechnology.”
Example 4:
defective (kharab) gene ko thik karna
isse genetic diseases treat ho sakti hain
Ye sab biotechnology ke parts hain
“The European Federation of Biotechnology (EFB) has given a definition…”
EFB (European Federation of Biotechnology) ne biotechnology ki definition di hai
“The definition given by EFB is as follows:”
Ab wo definition batayi ja rahi hai ,
“The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”
Explanation :
Natural science (biology, chemistry)
organisms (plants, animals, microbes)
cells ya unke parts
molecules (DNA, proteins)
in sab ko combine karke:
products (medicine, food)
services (healthcare, agriculture) banaye jaate hain.
Simple definition: Science + living things + molecules = useful products/services
9.1 Principles of Biotechnology
“Among many, the two core techniques that enabled birth of modern biotechnology are:”
Bahut techniques hain, lekin 2 main (core) techniques ne modern biotechnology ko janam diya :
i) Genetic engineering : Techniques to alter the chemistry of genetic material ( DNA and RNA ), to introduce these into host organisms and thus change the phenotype of the host organism.
Explanation : Aisi techniques (methods) jisse hum
DNA aur RNA ki structure/chemistry ko change karte hain.
Simple: Genes ko cut, copy, modify karna
Jo modified DNA banaya, usko
kisi host organism (jaise bacteria, plant, animal) ke andar daal dete hain
Simple: New gene ko kisi organism me insert karna.
Is gene insertion ki wajah se
us organism ka phenotype (dikhaai dene wale traits) change ho jata hai
Phenotype = appearance + behaviour + properties.
Summary : DNA ko modify karke usko kisi organism me daalna, jisse us organism ke traits (phenotype) change ho jaaye
Example:
Bacteria me human insulin gene daala
→ bacteria insulin banana start kar deta hai.
ii) Bioprocess engineering : Maintenance of sterile ( microbial contamination - free ) ambience in chemical engineering processes to enable growth of only the desired microbe / eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes,etc.
Explanation : Sterile ambience = esa environment jahan
koi unwanted microorganism (bacteria, fungi) nahi ho.
microbial contamination-free” ,means
unwanted germs se completely free
Simple: Lab/industry me bilkul clean, germ-free environment maintain karna.
ye sab kaam industrial level (factories/labs) me hota hai, jaha chemical engineering techniques use hoti hai.sirf vahi microorganism grow kare
jo ham chahte hai (desired microbe). unwanted microbes nahi hone chahiye nahi to ve product ko khrab kar denge.
Example:
Penicillin banane ke liye specific fungus hi grow hona chahiye.
us microbe ko bahut bari matra me grow karaana.
because industry me product mass level par banta hai. in microbes ka use karke
useful products banae jate hai.
Examples:
Antibiotics ( penicillin)
Vaccines (disease protection)
Enzymes (industrial & medical use)
Summary : Biotechnology me ham sterile environment maintain karte hai taki sirf desired microbe grow kare, use large quantity me badhakar antibiotics, vaccines, enzymes jaise products banae jaa sake.
Example : Penicillin production:
Fungus को sterile tank में grow किया जाता है
अगर contamination हो गया → पूरा batch खराब !
The basics of Genetic engineering
You probably appreciate the advantages of sexual reproduction over asexual reproduction. The former provides opportunities for variations and formulation of unique combinations of genetic setup,some of which may be beneficial to the organism as well as the population.Asexual reproduction preserves the genetic information,while sexual reproduction permits variation.
Explanation : Tum samajh sakte ho ki sexual reproduction, asexual reproduction se better hota hai, kyunki sexual reproduction mein do parents ka genetic material mix hota hai. Is mixing ki wajah se variation (differences) create hote hain, matlab har new organism thoda unique hota hai. Ye uniqueness isliye aati hai kyunki genes alag-alag combinations mein arrange hote hain—isse new genetic combinations bante hain.
Inmein se kuch variations aise hote hain jo organism ke liye faydemand hote hain—jaise better survival, disease resistance, ya environment ke according adjust karne ki ability. Jab ye beneficial traits kisi individual mein hote hain, to wo survive karke reproduce karta hai, aur dheere-dheere poori population mein wo trait spread ho jata hai.
Dusri taraf, asexual reproduction mein sirf ek parent hota hai aur offspring uska exact copy (clone) hota hai. Is process mein genetic information change nahi hoti, balki same hi rehti hai—matlab koi variation nahi aata.
Isliye, sexual reproduction variation allow karta hai, jo evolution aur long-term survival ke liye bahut important hai, jabki asexual reproduction sirf existing genetic information ko preserve karta hai.
Traditional hybridisation procedures used in plant and animal breeding,very often lead to inclusion and multiplication of undesirable genes along with the desired genes. The techniques of genetic engineering which include creation of recombinent DNA, use of genetic cloning and genetic transfer,overcome this limitation and allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.
Explanation : Traditional hybridisation (jo plant aur animal breeding mein use hota hai) ka matlab hota hai do alag parents ko cross karna taaki unke achhe traits (jaise high yield, disease resistance) ek saath aa jayein.
Lekin problem ye hai ki jab hum do organisms ko cross karte hain, to sirf achhe genes hi transfer nahi hote, balki unwanted (undesirable) genes bhi saath mein aa jate hain.
Kyunki genes chromosome par group mein hote hain, aur crossing ke time hum unhe perfectly separate nahi kar paate.
Isliye final offspring mein:
kuch desired traits aate hain
lekin saath hi kuch unwanted traits bhi aa jate hain.
Ye process controlled nahi hota, thoda random hota hai.
Ab genetic engineering aati hai, jo is problem ko solve karti hai.
Isme hum:
- DNA ko directly handle karte hain
- specific gene ko identify karte hain
- usko isolate (alag) karte hain.
Genetic engineering mein techniques hoti hain:
- recombinant DNA banana
- gene cloning (gene ki copies banana)
- gene transfer (target organism mein daalna)
Iska sabse bada advantage kya hai?
Hum sirf ek ya kuch selected desirable genes hi introduce karte hain, bina kisi unwanted gene ke.
Matlab:
traditional method = “mix sab kuch ho jata hai”
genetic engineering = “sirf jo chahiye wahi add karte hain”
Do you know the likely fate of a piece of DNA, which is somehow transferred into an alien organism ?
Most likely,this piece of DNA would not be able to multiply itself in the progeny cells of the organism.But , when it gets integrated into the genome of the recipient ,it may multiply and be inherited along with the host DNA.
Explanation : Socho agar koi DNA ka piece (foreign DNA) kisi dusre organism ke andar daal diya jaye, to uska kya hoga? Most cases mein, ye DNA apne aap survive ya multiply nahi kar pata. Iska reason ye hai ki DNA ko replicate hone ke liye proper signals aur sequence chahiye hote hain (jaise origin of replication), aur host cell ka system us foreign DNA ko usually recognize nahi karta.
Isliye jab cell divide karta hai, to ye DNA copy nahi hota aur dheere-dheere cells se gayab ho jata hai (lost ho jata hai).
Lekin agar wahi foreign DNA somehow host organism ke genome (chromosome) ke andar integrate ho jaye, to situation completely change ho jati hai. Ab wo DNA alag nahi raha, balki host DNA ka part ban gaya.
Ab jab bhi cell divide karega, host DNA ke saath-saath ye inserted DNA bhi copy hoga aur naye cells me chala jayega. Is tarah wo DNA stable ho jata hai aur generation to generation inherit bhi hota hai.
Origin of Replication :
This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate. In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ' origin of replication ' .
Explanation : Yeh isliye hota hai kyunki jo alien DNA (bahar ka DNA) hai, wo chromosome ka part ban jaata hai. Chromosome ek aisi structure hoti hai jisme DNA hota hai aur usme replicate (copy) hone ki ability hoti hai.
Chromosome ke andar ek special DNA sequence hota hai jise origin of replication kehte hain. Yeh ek starting point hota hai jahan se DNA ka replication (copy banana) start hota hai.
Isliye, agar kisi alien DNA ko organism ke andar multiply (zyada copies banana) karna hai, toh usse kisi chromosome ka part banna padega. Kyunki chromosome me hi origin of replication hota hai, jo replication ko start karta hai.
Simple line me: Alien DNA tabhi multiply karega jab wo chromosome ka part banega jisme origin of replication present ho.
Cloning :
This, an alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and multiply itself in the host organism. This can also be called as cloning or making multiple identical copies of any template DNA.
Explanation : alien DNA (bahar ka DNA) ko origin of replication ke saath link (jod) diya jata hai. Aisa isliye kiya jata hai taaki yeh alien DNA host organism ke andar replicate (copy) kar sake.
Jab alien DNA origin of replication se jud jata hai, toh cell ka replication system us DNA ko bhi copy karna start kar deta hai. Isse kya hota hai?
Ek DNA ki jagah uski bahut saari identical copies ban jaati hain.
Is process ko hi hum cloning kehte hain, matlab: kisi ek template DNA se uski same-to-same copies banana.
Simple line me: Alien DNA ko origin of replication ke saath jodne se wo host cell me replicate karta hai aur uski multiple identical copies ban jati hain — ise cloning kehte hain.
Construction of an artificial recombinant DNA molecule :
The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid ( autonomously replicating circular extra - chromosomal DNA ) of Salmonella typhimurium.Stanely Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. The cutting of DNA at specific locations became possible with the discovery of the source - called molecular scissors restriction enzymes.The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it. You probably know that mosquito acts as an insect vector to transfer the malarial parasite into human body.In the same way, a plasmid can be used as vector to deliver an alien piece of DNA into the host organism. The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA.
Explanation : “The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance…”
Hindieng :
Sabse pehla recombinant DNA tab bana jab scientists ne socha ki antibiotic resistance gene (jo bacteria ko dawa se bachata hai) ko kisi aur DNA ke saath joda ja sakta hai.
“…with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium.”
Hindieng :
Is gene ko ek plasmid ke saath joda gaya.
Plasmid kya hota hai?
Circular DNA hota hai.
Chromosome ke bahar hota hai (extra-chromosomal)
Apne aap replicate karta hai
Yeh plasmid bacteria Salmonella typhimurium ka tha.
“Stanley Cohen and Herbert Boyer accomplished this in 1972…”
Hindieng :
Yeh kaam 1972 me Stanley Cohen aur Herbert Boyer ne successfully kiya.
“…by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid…”
Hindieng :
Unhone plasmid se ek specific gene (antibiotic resistance) ko cut karke alag kiya.
“…which was responsible for conferring antibiotic resistance.”
Hindieng :
Yeh gene bacteria ko antibiotic se bachne ki ability deta hai.
“The cutting of DNA at specific locations became possible with the discovery of…”
Hindieng :
DNA ko exact jagah par cut karna ek discovery ke baad possible hua.
“…called molecular scissors restriction enzymes.”
Hindieng :
Is discovery ka naam hai restriction enzymes
Yeh DNA ko specific sites par cut karte hain
Isliye inhe molecular scissors kehte hain
“The cut piece of DNA was then linked with the plasmid DNA.”
Hindieng :
Jo DNA ka piece (gene) cut kiya gaya tha, use phir plasmid DNA ke saath jod diya gaya
“These plasmid DNA act as vectors to transfer the piece of DNA attached to it.”
Hindieng :
Yeh plasmid ek vector (carrier) ki tarah kaam karta hai
Hindieng : yeh attached DNA ko ek cell se dusre cell tak le jata hai
“You probably know that mosquito acts as an insect vector to transfer the malarial parasite into human body.”
Hindieng (example):
Jaise Mosquito malaria parasite ko human body me transfer karta hai, waise hi plasmid DNA ko transfer karta hai.
“In the same way, a plasmid can be used as vector to deliver an alien piece of DNA into the host organism.”
Hindeng :
Isi tarah plasmid ka use karke alien DNA (bahar ka DNA) ko host organism ke andar deliver kiya jata hai.
“The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase…”
Hindieng :
Gene aur plasmid ko jodne ke liye ek enzyme use hota hai — DNA ligase
“…which acts on cut DNA molecules and joins their ends.”
Hindeng :
DNA ligase ka kaam hai:
Cut DNA ke ends ko jodna (seal karna)
“This makes a new combination of circular autonomously replicating DNA created in vitro…”
Hindieng :
Is process se ek naya circular DNA banta hai jo lab me (in vitro) banaya gaya hai aur apne aap replicate kar sakta hai
“…and is known as recombinant DNA.”
Hindieng :
Is naye DNA ko hi recombinant DNA kehte hain.
Summary : Restriction enzyme se DNA cut hua → gene nikla → plasmid me insert hua → DNA ligase se join hua → naya DNA bana = recombinant DNA
When this DNA is transferred into Escherichia coli , a bacterium closely related to Salmonella ,it could replicate using the new host's DNA polymerase enzyme and make multiple copies.The ability to multiply copies of antibiotic resistance gene in E.coli was called cloning of antibiotic resistance gene in E.coli.
You can hence infer that there are three basic steps in genetically modifying an organism : ----
i) identification of DNA with desirable genes.
ii) introduction of the identified DNA into the host.
iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
Explanation : “When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella…”
Hindeng :
Jab yeh recombinant DNA bacteria Escherichia coli (E. coli) ke andar daala jata hai — jo Salmonella se related hai — tab process aage badhta hai.
“…it could replicate using the new host's DNA polymerase enzyme…”
Hindieng :
E. coli ke andar jaane ke baad yeh DNA, host ke DNA polymerase enzyme ki help se
apni copy banana (replicate hona) start kar deta hai.
“…and make multiple copies.”
Hindieng :
Is process se ek DNA ki bahut saari copies ban jaati hain.
“The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning…”
Hindieng :
Jab antibiotic resistance gene ki multiple identical copies ban jaati hain E. coli ke andar,
is process ko cloning kehte hain.
“…cloning of antibiotic resistance gene in E. coli.”
Hindieng :
Iska specific naam hai:
E. coli ke andar antibiotic resistance gene ki cloning
“You can hence infer that there are three basic steps in genetically modifying an organism:”
Hindieng :
Is poore process se hum yeh samajh sakte hain ki kisi organism ko genetically modify karne ke 3 main steps hote hain.
“i) identification of DNA with desirable genes.”
Hindieng :
Sabse pehle:
useful / desirable gene ko identify karna (jaise antibiotic resistance gene)
“ii) introduction of the identified DNA into the host.”
Hindieng :
Phir:
us gene ko host organism (jaise E. coli) ke andar daalna
. “iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.”
Hindieng :
Last step:
jo DNA dala gaya hai wo host ke andar stable rahe (maintain ho)
aur jab cell divide ho, toh yeh DNA next generation (progeny) me bhi transfer ho jaye
Definition
1.Recombinant DNA (rDNA):
Recombinant DNA is a DNA molecule formed by joining DNA fragments from two different sources, creating a new genetic combination.
2. Cloning:
Cloning is the process of producing multiple identical copies of a gene or DNA segment inside a host organism.
3. Plasmid:
A plasmid is a small, circular, double-stranded DNA molecule found in bacteria that can replicate independently of the chromosomal DNA and is used as a vector in genetic engineering.
4. Origin of Replication (ori):
Origin of replication is a specific DNA sequence from where replication of DNA begins, allowing the DNA to multiply inside the host cell.
5. Restriction Enzymes:
Restriction enzymes are molecular scissors that cut DNA at specific recognition sites, producing fragments of DNA.
6. DNA Ligase:
DNA ligase is an enzyme that joins two DNA fragments by forming phosphodiester bonds between them.
9.2 Tools of Recombinant DNA Technology
“Genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools…”
Hinglish: Genetic engineering (DNA ko modify karna) tabhi possible hai jab hamare paas kuch zaroori tools ho.
“…restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.”
Yeh tools hain:
- restriction enzymes (eeDNA kaatne ke liye)
- polymerase (DNA banane ke liye)
- ligase (DNA jodne ke liye)
- vector (DNA carry karne ke liye)
- host organism (jisme DNA daalte hain)e
9.2.1 Restriction Enzymes
In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated.One of these added methyl groups to DNA, while the other cut DNA.The later was called restriction endonuclease.
Explanation : 1963 mein, E. coli bacteria ke andar virus (bacteriophage) ki growth ko restrict(kam) karne wale do enzymes ko isolate(alag) kiya gaya. Inmein se ek enzyme DNA par methyl groups add karta tha, aur doosra DNA ko cut karta tha. Baad wale (yaani kaatne wale) enzyme ko restriction endonuclease kaha gaya.
The first restriction endonuclease—Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later.It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six based pairs.This specific based sequence is known as the recognition sequence for Hind II. Besides Hind II , today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.
Explanation : Sabse pehla restriction enzyme Hind II tha, jise scientists ne 5 saal baad isolate karke study kiya. Is enzyme ki khas baat yeh hai ki yeh randomly kaam nahi karta, balki DNA ke ek specific nucleotide sequence par hi kaam karta hai. Yeh hamesha DNA ko ek fixed jagah par cut karta hai, kyunki yeh 6 base pairs ke ek particular sequence ko pehchanta hai. Is special sequence ko recognition sequence kaha jata hai, jo basically enzyme ke liye ek “target spot” hota hai jahan wo cut karta hai. Hind II ke alawa, aaj ke time me 900 se bhi zyada restriction enzymes discover ho chuke hain, jo 230 se zyada bacteria ke different strains se mile hain, aur har enzyme apna alag-alag recognition sequence pehchanta hai aur usi jagah DNA ko cut karta hai.
The convention for naming these enzymes is the first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated,e.g. EcoR1 comes from Escherichia coli RY 13. In EcoR1 ,the letter 'R' is derived from the name of strain.Roman numbers following the names indicate the order in which the enzymes were isolated from the strain of bacteria.
Explanation : Restriction enzymes ka naam rakhne ka ek fixed rule hota hai. Naam ka pehla letter us bacteria ke genus se liya jata hai, aur agle do letters uske species se aate hain jisse enzyme nikala gaya hai. Jaise EcoRI naam Escherichia coli se aaya hai. Isme “E” genus (Escherichia) se, aur “co” species (coli bacteria or prokaryotic cell ) se liya gaya hai. Iske baad jo “R” hai wo us particular strain (RY13) ka naam show karta hai, jisse enzyme isolate kiya gaya tha. Aur jo Roman number “I” hai, wo batata hai ki us strain se yeh pehla enzyme isolate hua tha (agar II, III hota to matlab second, third enzyme hota).
E = Escherichia (Genus)
co = coli (Species)
R = RY 13 (Strain)
I = First discovered (Pehla discovery
Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds : exonuclease and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
Each restriction endonuclease functions by inspecting the length of a DNA sequence. Once it finds its specific recognition sequence,it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar - phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
Explanation : Restriction enzymes ek bade group ka part hote hain jise nucleases bolte hain, yani aise enzymes jo DNA ko cut karte hain. Nucleases do type ke hote hain—exonucleases aur endonucleases. Exonucleases DNA ke ends (shuru ya last) se nucleotides ko hataate hain, matlab wo DNA ko dheere-dheere bahar se chhota karte hain. Jabki endonucleases DNA ke andar (beech me) kisi specific jagah par cut lagate hain. Ab restriction endonucleases special type ke endonucleases hote hain jo DNA ko randomly nahi, balki ek specific sequence par hi cut karte hain.
Ye enzymes DNA ko scan karte rehte hain, jaise kisi word ko dhund rahe ho. Jaise hi unhe apna recognition sequence milta hai, wo DNA se chipak (bind) jate hain aur double helix ki dono strands ko ek exact jagah par cut kar dete hain. Ye cut DNA ke sugar-phosphate backbone me hota hai (jo DNA ka outer structure hota hai). Sabse important baat ye hai ki ye enzymes ek palindromic sequence ko pehchante hain, matlab aisa sequence jo dono taraf se same read hota hai (jaise “GAATTC” ko ulta padho to bhi same pattern milta hai). Isi wajah se ye enzymes DNA ko bilkul exact aur predictable jagah par cut kar pate hain, jo genetic engineering me bahut useful hota hai.
Do you know what palindromes are ?
These are groups of letters that form the same words when read both forward and backward,e.g.. " MALAYALAM ". As against a word - palindrome where the same word is ready in both directions,the palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
For example, the following sequences reads the same on the two strands in 5' ------ 3' direction. This is also true if read in the 3' -------- 5' direction.
5' ------ GAATTC -------- 3'
3' -------- CTTAAG --------5' 
Explanation : Palindrome wo letters ya words hote hain jo aage se aur peeche se same read hote hain, jaise “MALAYALAM”. Matlab agar tum ise left se right ya right se left padho, dono case me word same hi rahega. Lekin DNA me palindrome thoda different hota hai. Yahan ek hi word ko reverse nahi padhte, balki DNA ke do strands (double helix) ko consider karte hain. DNA palindrome ka matlab hota hai ki base pair sequence dono strands par same dikhe jab hum unhe same direction (5' → 3') me padhen.
DNA me bases complementary hote hain (A-T aur G-C), isliye dono strands identical nahi hote, par phir bhi special arrangement ke wajah se palindrome ban sakta hai. Jaise example me diya hai:
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
Agar hum pehle strand ko 5' → 3' me padhen to sequence “GAATTC” milta hai. Ab second strand ko bhi agar same direction (5' → 3') me padhna hai, to hume usse reverse karna padega. Jab hum “CTTAAG” ko reverse karte hain, to wo “GAATTC” ban jata hai. Is tarah dono strands ko same direction me padhne par same sequence milta hai, isliye ye DNA palindrome kehlaata hai.
Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two based on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
Explanation : Restriction enzymes DNA ko cut karte hain, lekin ye cut bilkul palindrome sequence ke exact center par nahi hota. Ye enzymes thoda sa center se hatkar (away from centre) cut lagate hain, aur dono strands par same type ke bases ke beech me cut karte hain (staggered cut). Is wajah se DNA ke ends bilkul seedhe (straight) nahi bante, balki chhote-chhote single stranded portions (overhangs) bach jaate hain.
In overhanging parts ko “sticky ends” kaha jata hai. Inhe sticky isliye bolte hain kyunki ye easily apne complementary base sequences ke saath hydrogen bonds bana lete hain. Matlab agar kisi doosre DNA fragment ke ends matching (complementary) ho, to ye ends aapas me chipak jaate hain.
Ye “stickiness” bahut important hoti hai, kyunki isi ki help se enzyme DNA ligase kaam karta hai. DNA ligase in joined fragments ko permanently jod deta hai, yani sugar-phosphate backbone ko seal kar deta hai. Is tarah alag-alag DNA fragments ko jodna easy ho jata hai.
Restriction endonucleases are used in genetic engineering to form ' recombinant ' molecules of DNA, which are composed of DNA from different sources/ genomes.
When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ' sticky - ends ' and, these can be joined together ( end - to - end ) using DNA ligases. You may have realised that normally, unless one cuts the vector and the source DNA with the same restriction enzyme,the recombinant vector molecule cannot be created.
Explanation : Restriction endonucleases genetic engineering me use kiye jaate hain recombinant DNA molecules banane ke liye. Recombinant DNA ka matlab hota hai aisa DNA jo alag-alag sources ya genomes se aaya ho aur jod diya gaya ho (mix DNA).
Jab hum DNA ko same restriction enzyme se cut karte hain, to jo DNA fragments bante hain un sabke ends same type ke “sticky ends” hote hain. Ye sticky ends ek dusre ke complementary hote hain, isliye ye easily aapas me jud jaate hain (end-to-end pairing). Iske baad enzyme DNA ligase in fragments ko permanently join kar deta hai.
Ek important baat: normally recombinant DNA tabhi banega jab vector DNA (jaise plasmid) aur source DNA (jisme desired gene hai) dono ko same restriction enzyme se cut kiya jaaye. Agar alag-alag enzymes use karoge, to sticky ends match nahi karenge, aur DNA fragments properly join nahi honge. Isliye recombinant vector molecule banana possible nahi hoga.
Separation and Isolation of DNA fragments
Separation = sab fragments ko alag-alag line (bands) me lana.
Isolation = unme se ek specific fragment ko nikaal lena.
Jab DNA ko Restriction Endonuclease se cut kiya jata hai, to DNA chhote-chhote tukdon (fragments) me break ho jata hai. Lekin ye saare fragments ek hi solution me mix rehte hain, isliye inhe alag karna zaroori hota hai. In fragments ko size ke hisaab se separate karne ke liye ek technique use hoti hai jise Gel Electrophoresis kehte hain.
DNA fragments negative charged hote hain, isliye jab electric current diya jata hai to ye + charge (anode) ki taraf move karte hain. Ye movement ek gel ke andar hoti hai jise matrix kehte hain. Aajkal sabse commonly agarose gel use hota hai, jo seaweed se milta hai.
Agarose gel ek jaali (sieve) ki tarah kaam karta hai. Jab DNA fragments is gel ke through move karte hain, to ye apne size ke hisaab se alag ho jaate hain. Bade DNA fragments gel ke pores me atak jaate hain, isliye dheere move karte hain aur wells ke paas hi reh jaate hain. Chhote DNA fragments aasani se gel ke pores se nikal jaate hain, isliye tezi se move karte hain aur zyada door tak pahunch jaate hain. Isi wajah se rule hota hai: jitna chhota fragment, utna zyada door move karega.
Is process ke baad DNA fragments alag-alag bands ke form me milte hain, lekin DNA directly visible nahi hota. Isliye DNA ko dekhne ke liye use ek dye se stain kiya jata hai jaise Ethidium Bromide, aur phir UV light me dekha jata hai jahan ye bright orange color ke bands ke roop me dikhta hai. Har band same size ke DNA fragments ko represent karta hai.
Agar kisi specific DNA fragment ki zarurat ho, to us band ko gel se cut karke DNA ko nikaala jata hai. Is process ko elution kehte hain. Is tarah se purified DNA fragments ko baad me recombinant DNA banane ke liye use kiya jata hai.
Gel electrophoresis : The cutting of DNA by restriction endonucleases results in the fragments of DNA.These fragments can be separated by a technique known as gel electrophoresis.
Elution : The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.
 | ||||
| Lane 1 (Undigested): Isme DNA ko kata nahi gaya hai, isliye wo ek bada band banakar wells ke paas hi rehta hai. | Lanes 2-4 (Digested): Isme DNA ko enzymes se fragments mein kaat diya gaya hai, isliye alag-alag bands dikh rahe hain. | Movement Rule: DNA negative charge hota hai, isliye wo positive side ki taraf move karta hai. | Size Factor: Jo fragment sabse chhota (Smallest) hota hai wo sabse door tak jata hai, aur bada (Largest) fragment wells ke paas rehta hai. |
9.2.2 Cloning Vectors
You know that plasmid and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.Some plasmids may have only one or two copies per cell whereas others may have 15 - 100 copies per cell. Their numbers can go even higher. If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA,we can multiply its numbers equal to the copy number of the plasmid or bacteriophage. Vectors used at present,are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non - recombinants.
Explanation : Plasmid aur bacteriophage aise DNA elements hote hain jo bacterial cell ke andar chromosomal DNA ke control ke bina hi apne aap replicate (copy) ho sakte hain. Iska matlab ye independent hote hain aur baar-baar multiply karte rehte hain. Bacteriophages ka number ek cell ke andar kaafi zyada hota hai, isliye unke genome ki copies bhi bahut high hoti hain. Plasmids me variation hota hai—kuch plasmid sirf 1–2 copies per cell me hote hain (low copy), jabki kuch 15–100 ya usse bhi zyada copies bana sakte hain (high copy).
Ab agar hum kisi foreign ya alien DNA ko plasmid ya bacteriophage ke DNA ke saath jod dete hain, to wo foreign DNA bhi unke saath-saath utni hi baar copy ho jata hai jitna plasmid ya phage replicate hota hai. Isi wajah se ye genetic engineering me bahut useful hote hain, kyunki ye foreign DNA ko multiply kar dete hain.
Aaj kal jo vectors use kiye jate hain (jaise modified plasmids ya phages), unhe specially engineer kiya jata hai taaki foreign DNA ko easily insert (link) kiya ja sake aur baad me hum easily identify kar saken ki kaun se cells me foreign DNA successfully insert hua hai (recombinant) aur kaun se me nahi (non-recombinant).
Engineered = natural cheez ko modify karke usko kaam ke liye perfect banana.
The following are the features that are required to facilitate cloning into a vector.
Hindieng : Ek vector ke andar cloning ko asaan banane ke liye ye neeche di gayi cheezein hona bahut zaroori hain.
(i) Origin of replication (ori):
Line: This is a sequence from where replication starts...
Hindieng : Ye DNA ka wo khaas tukda hai jahan se photocopy (replication) shuru hoti hai.
Line: ...and any piece of DNA when linked to this sequence can be made to replicate within the host cells.
Hindieng : Agar hum kisi bhi bahar ke DNA ko is 'ori' se jod dein, to wo host cell ke andar apni copies banana shuru kar dega.
Line: This sequence is also responsible for controlling the copy number of the linked DNA.
Hindieng : Ye 'ori' hi decide karta hai ki jo DNA humne joda hai, uski kitni copies (kam ya zyada) banengi.
Line: So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.
Hindieng : Isliye, agar tumhe apne DNA ki bahut saari copies chahiye, to use aise vector mein daalo jiska 'ori' zyada copy number banane mein madad kare.
(ii) Selectable marker:
Line: In addition to ‘ori’, the vector requires a selectable marker...
Hindieng : 'ori' ke saath-saath vector mein ek 'selectable marker' bhi hona chahiye.
Line: ...which helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants.
Hindieng : Iska kaam hai un cells ko pehchan kar khatam kar dena jinme naya DNA nahi gaya (non-transformants), aur sirf sahi waale cells (transformants) ko badhne dena.
Line: Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
Hindieng : Transformation wo tarika hai jisse hum DNA ke tukde ko bacteria ke andar ghusate hain.
Line: Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli.
Hindieng : Wo gene jo bacteria ko ampicillin ya tetracycline jaisi dawaiyon se bachate hain, unhe hi best marker mana jata hai.
Line: The normal E. coli cells do not carry resistance against any of these antibiotics.
Hindieng : Jo sadharan E. coli hota hai, uske paas in dawaiyon se bachne ki koi shakti nahi hoti (wo mar jata hai).
iii) Cloning sites :
Line: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
HindiEnglish: Agar bahar ka DNA (alien DNA) vector se jodna hai, to vector mein enzyme ke kaatne wali jagah (recognition sites) bahut kam honi chahiye. Ho sake to ek enzyme ke liye sirf ek hi site ho.
Line: Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning (Figure 9.4).
HindiEnglish: Agar vector mein ek hi enzyme ki bahut saari sites hongi, to enzyme use kai jagah se kaat dega aur bahut saare tukde (fragments) ho jayenge. Isse cloning ka kaam bahut mushkil (complicate) ho jayega.
Line: The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
HindiEnglish: Naya DNA hum vector ke un do genes (amp^R ya tet^R) mein se kisi ek ke andar jaakar jodte hain jo antibiotic se bachate hain.
Line: For example, you can ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322.
HindiEnglish: Jaise ki, tum pBR322 vector mein tetracycline resistance gene (tet^R) ke andar jo BamH I wali site hai, wahan apna DNA jod sakte ho.
Line: The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA...
HindiEnglish: Jab tum naya DNA ghusaoge, to wo tetracycline wala gene toot jayega aur apni shakti kho dega (lose resistance). Ab ye bacteria tetracycline dawa se nahi bach payega.
Line: ...but can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium.
HindiEnglish: Iske bawajood, hum ise un bacteria se alag pehchan sakte hain jinme DNA nahi juda (non-recombinants), bas humein inhe tetracycline wali plate par ugana hoga.
Line: The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
HindiEnglish: Pehle hum saare transformants ko ampicillin wali plate par ugate hain, fir wahan se utha kar unhe tetracycline wali plate par rakhte hain check karne ke liye.
Line: The recombinants will grow in ampicillin containing medium but not on that containing tetracycline.
HindiEnglish: Jo recombinants honge (jinme naya DNA jud gaya hai), wo ampicillin par to zinda rahenge, lekin tetracycline par mar jayenge (kyunki unka tet^R gene tumne tod diya hai).
Line: But, non-recombinants will grow on the medium containing both the antibiotics.
HindiEnglish: Lekin non-recombinants (jinme naya DNA nahi juda) dono dawaiyon (ampicillin aur tetracycline) par maze se zinda rahenge kyunki unke dono gene sahi-salamat hain.
Line: In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated’ due to insertion of alien DNA, and helps in selection of recombinants.
HindiEnglish: Is poore process mein, ek gene transformants pehchanne mein madad karta hai, aur doosra gene (jo naya DNA judne se bekar ya inactivated ho gaya hai) wo recombinants ko pehchanne mein madad karta hai.
Screenshot 2: Page 170 (Selection ka Asaan Tarika)
Line: Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics.
HindiEnglish: Antibiotic ki madad se recombinants ko chunna bahut jhanjhat wala (cumbersome) kaam hai kyunki isme do alag-alag plates par baar-baar bacteria ko ugana aur check karna padta hai.
Line: Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
HindiEnglish: Isliye scientists ne doosre markers dhoondhe jo rang (colour) ke aadhar par recombinants aur non-recombinants ke beech antar bata sakein. Isme ek khaas chemical (chromogenic substrate) ka use hota hai.
Line: In this, a recombinant DNA is inserted within the coding sequence of an enzyme, \beta-galactosidase.
HindiEnglish: Is naye tarike mein, hum apna naya DNA \beta-galactosidase enzyme banane wale gene ke beech mein daal dete hain.
Line: This results into inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation.
HindiEnglish: Isse enzyme banane wala gene bekar ho jata hai. Is process ko hum 'insertional inactivation' kehte hain.
Line: The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert.
HindiEnglish: Agar bacteria ke plasmid mein naya DNA nahi juda hai, to enzyme banega aur chemical ki wajah se colony ka rang Blue (neela) dikhega.
Line: Presence of insert results into insertional inactivation of the \beta-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.
HindiEnglish: Agar naya DNA (insert) jud gaya hai, to enzyme nahi banega aur colony ka koi rang nahi aayega (wo safed rahegi). In safed colonies ko hi hum recombinant kehte hain.
iv) Vectors for Cloning genes in plants and animals :
Line: You may be surprised to know that we have learnt the lesson of transferring genes into plants and animals from bacteria and viruses which have known this for ages...
HindiEnglish: Aapko jaan kar hairani hogi ki paudhon (plants) aur janwaro (animals) mein gene kaise transfer karte hain, ye humne un bacteria aur viruses se seekha hai jo ye kaam sadiyon se karte aa rahe hain.
Line: ...how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want.
HindiEnglish: Wo bacteria/viruses jaante hain ki eukaryotic cells (plants/animals) ke andar gene kaise daalna hai aur unse wo kaam kaise karwana hai jo bacteria ya virus chahte hain.
Line: For example, Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor...
HindiEnglish: Jaise ki, Agrobacterium tumifaciens naam ka bacteria bahut saare dicot plants ke liye ek bimari (pathogen) hai. Ye bacteria apna ek DNA ka tukda, jise 'T-DNA' kehte hain, paudhe ke cell mein daal deta hai aur normal cells ko tumor (cancerous cells) mein badal deta hai.
Line: ...and direct these tumor cells to produce the chemicals required by the pathogen.
HindiEnglish: Aur ye tumor cells fir wahi chemicals banane lagte hain jo us bacteria (pathogen) ko apne khaane ya rehne ke liye chahiye hote hain.
Line: Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.
HindiEnglish: Bilkul isi tarah, janwaro mein retroviruses (jaise HIV ka khandaan) normal cells ko cancer waale cells mein badalne ki taqat rakhte hain.
Line: A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these expression vectors...
HindiEnglish: Jab humein samajh aa gaya ki ye bacteria aur virus gene transfer karne ki is kala (art) mein kitne mahir hain, to humne isi knowledge ka use karke inhe expression vectors mein badal diya.
Line: ...into useful vectors for delivering genes of interest to humans.
HindiEnglish: Taaki ab ye bimari failane ki jagah hamare kaam ke genes (genes of interest) ko cells tak pahunchane ka kaam karein.
Line: The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants...
HindiEnglish: Agrobacterium ka wo Ti plasmid (jo tumor banata tha), ab humne aise badal diya hai (modify kar diya hai) ki wo ab paudhon ko bimar nahi karta (no more pathogenic).
Line: ...but is still able to use the mechanisms to deliver genes of our interest into a variety of plants.
HindiEnglish: Lekin uske andar abhi bhi wo machine/power hai jisse wo hamare pasand ke genes ko alag-alag paudhon mein deliver kar sake.
Line: Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.
HindiEnglish: Waise hi, retroviruses ko bhi humne 'disarm' kar diya hai (unke hathiyar yaani bimari failane wale gene nikal diye hain). Ab wo janwaro ke cells mein acche genes pahunchane ke kaam aate hain.
Line: So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).
HindiEnglish: To jab ek baar hamara gene kisi sahi vector (Agrobacterium ya Retrovirus) ke saath jud (ligate) jata hai, tab use host cell (bacteria, plant ya animal) mein daal diya jata hai jahan wo apni sankhya badhata hai.
Naye Terms ka Matlab (Short Summary):
Pathogen: Bimari failane wala jeev (bacteria/virus).
T-DNA: Agrobacterium ka wo DNA jo tumor banata hai.
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| Figure.9.4 PBR322 |
Ti Plasmid: 'Tumor Inducing' plasmid.
Disarmed: Virus ya bacteria ki bimari failane ki shakti ko nikal dena.
Host: Wo cell jiske andar hum naya DNA bhej rahe hain.
Figure 9.4: pBR322 Diagram explanation :
p stands for: Plasmid (Bacteria ka circular DNA)
B stands for: Bolivar (Scientist ka naam).
R stands for: Rodriguez (Doosre scientist ka naam).
322 stands for: Number (Laboratory mein diya gaya code).
1. ori (Origin of Replication)
Function: Ye wo "Start Button" hai jahan se DNA apni copy banana shuru karta hai. Agar kisi bahar waale DNA ko is vector mein zinda rehna hai aur apni sankhya badhani hai, to use ori se judna hi padega.
Extra Point: Ye "Copy Number" ka maalik hai. Matlab ek cell mein 20 copy banengi ya 100, ye ori hi tay karta hai.
2. rop (Repressor of Primer)
Term: rop gene.
Function: Iska kaam hai aise proteins banana jo replication ki speed ko control karein.
Cutting Site: Iske paas Pvu II naam ka cutting point hota hai.
Function of Pvu II : DNA ko kaat kar naya gene jodne ki jagah dena.
3. Antibiotic Resistance Genes (Selectable Markers)
Ye dono bacteria ke liye "Suraksha Kavach" hain:
ampᴿ (Ampicillin Resistance):
Function : Ye gene bacteria ko Ampicillin (ek antibiotic dawa) se marne nahi deta.
Cutting Points: Iske andar do gates hain—Pst I aur Pvu I. Agar tumne yahan naya DNA ghusaya, to Ampicillin wala suraksha kavach toot jayega.
tetᴿ (Tetracycline Resistance):
Function : Ye gene bacteria ko Tetracycline dawa se bachata hai.
Cutting Points: Iske andar BamH I aur Sal I sites hain. Agar yahan naya DNA dala, to Tetracycline wala kavach bekar ho jayega.
4. Restriction Enzyme Sites (The Scissors)
Ye wo points hain jahan "Biological Scissors" (enzymes) cut lagate hain. Diagram mein jo baki naam hain wo enzymes ke hain:
EcoR I, Cla I, Hind III: Ye teeno vector ke oopari hisse mein hain. Yahan kaat kar DNA jodne se kisi bhi antibiotic gene ko nuksan nahi pahunchta.
BamH I / Sal I: Ye tetᴿ ke dushman hain.
Pst I / Pvu I: Ye ampᴿ ke dushman hain.
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