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Structure and Types of RNA:-Basic Structure of RNA:- The ribonucleic acid has all the components same to that of the DNA with only 2 main differences within it-
i. Thymine which is replaced by the uracil.
ii. Deoxyribose sugar is replaced by Ribose sugar.
Types of RNA:-
i. mRNA:- It is transcribed from DNA and contains the genetic information to make proteins. It has a start codon and a stop codon for protein synthesis. Prokaryotic mRNA is simple. Eukaryotic mRNA has a 5' cap and 3' poly A tail. The 5’ cap protects the mRNA from degradation, and the 3’ poly(A) tail contributes to the stability of mRNA and aids it in transport.
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ii. tRNA:- These are RNA molecules that translate mRNA into proteins. They of a 3’ acceptor site, 5’ terminal phosphate, D arm, T arm, and anticodon arm. The primary function of a tRNA is to carry amino acids on its 3’ acceptor site to a ribosome complex with the help of aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases are enzymes that load the appropriate amino acid onto a free tRNA to synthesize proteins. Once an amino acid is bound to tRNA, the tRNA is considered an aminoacyl-tRNA. tRNA has an L shaped 3D structure. It is specific to each amino acid and carries an amino acid to the growing chain of a polypeptide during the translation process.
Secondary Structure:- It has a shape similar to Clover leaf.
Tertiary Structure:- It has a shape similar to Inverse L letter.
iii. rRNA:- rRNA forms ribosomes, which are essential in protein synthesis. A ribosome contains a large and small ribosomal subunit. In prokaryotes, a small 30S and large 50S ribosomal subunit make up a 70S ribosome. In eukaryotes, the 40S and 60S subunit form an 80S ribosome. The ribosomes contain an exit (E), peptidyl (P), and acceptor (A) site to bind aminoacyl-tRNAs and link amino acids together to create polypeptides.
iv. Small nuclear RNAs (snRNA):- These are non-coding RNAs that are responsible for splicing introns. The snRNAs join with proteins to form small nuclear ribonucleoproteins (snRNP), which most commonly contain U1, U2, U4, U5, and U6 snRNA molecules.
v. MicroRNA (miRNA):- These are non-coding RNAs mainly involved in gene regulation. They are mostly processed from introns and are transcribed into primary miRNA from the host gene by RNA polymerase II.
vi. Small Interfering RNAs (siRNA):- These are double-stranded, non-coding RNAs that inhibit gene expression through RNA interference. They interfere with gene expression by degrading mRNA and preventing the translation of proteins.
Transcription:-
Introduction:-
• It is the process of copying genetic information from one strand of the DNA into RNA.
• Here, adenine pairs with uracil instead of thymine.
• Both strands are not copied during transcription, because:-
- The code for proteins is different in both strands. This complicates the translation.
- If 2 RNA molecules are produced simultaneously this would be complimentary to each other, hence form a double stranded RNA. This prevents translation.
Transcription Unit:-
• It is the segment of DNA between the sites of initiation and termination of transcription. It consists of 3 regions:-
- A promoter (Transcription start site):- Binding site for RNA polymerase.
- Structural gene:- The region between promoter and terminator where transcription takes place.
- A terminator:- The site where transcription stops.
• The DNA- dependent RNA polymerase catalyzes the polymerization only in 5’→3’direction.
• 3’→5’ acts as template strand and 5’→3’ acts as coding strand.
3’-ATGCATGCATGCATGCATGCATGC-5’ template strand.
5’-TACGTACGTACGTACGTACGTACG-3’ coding strand.
Transcription unit and gene:-
• Gene:- Functional unit of inheritance. It is the DNA sequence coding for RNA molecule.
• Cistron:- A segment of DNA coding for a polypeptide.
• Structural gene in a transcription unit is of 2 types:-
i. Monocistronic structural genes (split genes):- It is seen in eukaryotes. Here, the coding sequences exons are interrupted by introns.
ii. Polycistronic structural genes:- It is seen in prokaryotes. Here, there are no split genes.
Reverse Transcription:- It is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses, such as the human immunodeficiency virus (HIV).
Steps of transcription in prokaryotes:-
i. Initiation:- Here, the enzyme RNA polymerase binds at the promoter site of DNA. This causes the local unwinding of the DNA double helix. An initiation factor (σ factor) present in RNA polymerase initiates the RNA synthesis.
ii. Elongation:- The RNA chain is synthesized in the 5’-3’ direction. In this process, activated ribonucleoside triphosphates (ATP, GTP, UTP & CTP) are added. This is complementary to the base sequence in the DNA template.
iii. Termination:- A termination factor (ρ factor) binds to the RNA polymerase and terminates the transcription.
NOTE:- In bacteria (Prokaryotes) transcription and translation can be coupled (Translation can begin before mRNA is fully transcribed) because:-
- mRNA requires no processing to become active.
- Transcription and translation take place in the same compartment (no separation of cytosol and nucleus).
Polysome or Polyribosome or Ergosome:- It is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.
Eukaryotic transcription:-
In eukaryotes, there are 2 additional complexities-
i. There are 3 RNA polymerases:-
• RNA polymerase I:- Transcribes rRNAs (28S, 18S & 5.8S).
• RNA polymerase II:- Transcribes mRNA.
• RNA polymerase III:- Transcribes tRNA, 5S rRNA and snRNAs.
ii. The primary transcripts (hnRNA):- It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes splicing process.
Control at Initiation:-
i. Steric hindrance for RNA polymerase binding:- If the operator and – 35 sequence overlap, repressor could prevent the formation of a closed complex by preventing polymerase binding. It is also possible that repressor binding can prevent the activator's interaction with polymerase and/or DNA.
ii. Inhibition of open complex formation:- Isomerization is a step in which RNA polymerase conformation is changed and a more stable complex is formed spanning the region of + 5 to beyond – 35 sequence. The exact boundary of nucleotides protected by RNA polymerase in footprinting
experiments varies depending on the promoter used in an experiment. The repressor when bound at or near the – 10 region of the promoter (the site of complex formation) could distort the DNA and as a consequence, formation of stable open complexes may be prevented. The Arc repressor involved in bacteriophage P22 lysogeny is believed to repress the Pant promoter in this fashion.
iii. Inhibition of initiation complex formation:- RNA polymerase initiates RNA synthesis with the formation of nascent RNA of 3-8 nucleotides. During this stage, polymerase is still bound to the promoter. The repressor which is bound to the operator may make direct contacts with the enzyme present in open complex state, thus preventing the subsequent step, i.e. oligoribonucleotide formation. Studies with the gal repressor support this mechanism. The gal repressor does not block open complex formation at the gal operon promoter and hence is believed to interfere at the stage of oligoribonucleotide formation.
iv. Increased abortive initiation:- Once an initiation complex is formed, RNA polymerase moves out of the promoter and gets locked into the elongation mode. The repressor could make contacts with RNA polymerase and block promoter clearance.
Control at termination:-
Attenuation:- Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.
i. Transcriptional-attenuation:- It is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.
ii. Translational-attenuation:- It is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.
Heterogenous RNA Processing:- The hnRNA is the collective term for the unprocessed mRNA (pre-mRNA) molecules in the nucleus. It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes the following processes-
i. Splicing:- From hnRNA introns are removed by the spliceosome and exons are joined together.
ii. Capping:- Here, a nucleotide methyl guanosine triphosphate (cap) is added to the 5’ end of hnRNA.
iii. Tailing (Polyadenylation):- Here, adenylate residues (200-300) are added at 3’-end. It is the fully processed hnRNA, now called mRNA.
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Blotting:-
> It is the separation and transfer of macromolecules onto a blotting membrane to detect and identify such macromolecules, as DNA, RNA or Protein.
> 4 types:-
i. Southern blotting:- Detect DNA.
ii. Northern blotting:- Detect RNA.
iii. Western blotting:- Detect Protein.
iv. Eastern blotting:- Detect Protein.
General Procedure:-
- The target molecule in a sample is isolated.
- Electrophoresis separates the molecules.
- The separated contents are transferred onto a membrane. This process is celled blotting.
- The membrane is then exposed to radiolabeled probes and incubated. Probe binds to the target molecule.
- The probe and target molecule create bands that can be visualized with X-ray film.
1. Southern blotting:-
- Discovered by Edward M. Southern at Edinburgh University in 1970.
- It is designed to locate a particular sequence of DNA within a complex mixture. For example, it could be used to locate a particular gene within an entire genome.
Procedure:-
a. Digestion:- Digest the DNA with an appropriate restriction enzyme.
b. Electrophoresis:- Run the digest on an agarose gel.
c. Denaturation:- Denature the DNA on the gel. For this soak it in about NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.
d. Depurination:- Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl takes the purines out, cutting the DNA into smaller fragments. Neutralize the acid after this step.
e. Blotting:-
- It is the transfer the denatured DNA to the membrane.
- A nitrocellulose membrane or nylon membrane is used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm.
- Many scientists feel nylon is better since it binds more and is less fragile.
- Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.
- You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC (Saline Sodium Citrate) through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)
- After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)
f. Hybridization:-
- It is the process of annealing the probe to the DNA on the membrane due to complementarity.
- Probing is often done with:
i. 32P labeled ATP
ii. Biotin / Streptavidin
iii. Bioluminescent probe
g. Visualization:-
- Visualize your radioactively labeled target sequence.
- If you used a radiolabeled 32P probe, then you would visualize by autoradiograph.
- Biotin / Streptavidin detection is done by colorimetric methods.
- Bioluminescent visualization uses luminesence.
2. Northern Blotting:-
- The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
- Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.
- It is designed to locate a particular sequence of RNA within a complex mixture.
Procedure:-
a. Extraction of RNA:- There are many RNA extraction kits commercially available, but they all involve cell lysis, inhibition of RNAases, removal of proteins and other contaminants, and recovery of RNA.
b. Isolation of mRNA:-
- Oligo dT cellulose chromatography can be used to isolate only mRNA with a polyA tail. The poly A tail is the final step of mRNA production in the nucleus. The tail enables nuclear export, translation, and stability of mRNA.
- In Oligo dT cellulose chromatography, oligos complementary to the poly A tail are covalently attached to a resin column.
- When the sample is applied to the column the mRNA with the poly A tail will hybridize to the oligo probe and be retained on the column.
- Then, the elution buffer is applied to disrupt hybridization and recover the mRNA.
c. Gel electrophoresis to separate mRNA by size:-
- Agarose gels containing formaldehyde were traditionally used to denature RNA.
- The formaldehyde reacts with the imine and amine groups on the nucleic acids, which disrupts the hydrogen bonding between bases and disrupts the secondary structure of the RNA.
- It is important to disrupt the secondary structure because the RNA must be extended to allow proper binding of probe for identification.
d. Transfer of RNA to blotting membrane:-
- The transfer is necessary because the probes can’t enter into the gel matrix. Therefore, the RNA must be transferred to a membrane where they can be accessed by the probes.
- Transfer is accomplished via a capillary (overnight) or vacuum (15-60 minutes) blotting system.
- The blotting membrane is positively charged to attract the negatively charged RNA. Nylon is a commonly used membrane.
v. Immobilization of RNA to the blotting membrane:- Covalently attached to the membrane by the application of UV light or heat.
vi. Application of Probe:-
- Probes have a minimum of 25 bases that are complimentary to the mRNA sequence of interest.
- Excess probe is washed off.
vii. Probe visualization:-
- Radioactive isotopes were traditionally used, but have been replaced in favor of safer detection methods.
- Chemiluminescence is commonly used in the modern northern blot protocol.
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Reverse transcriptase:- It is an RNA-dependent DNA polymerase—an enzyme that synthesizes complementary DNA (cDNA) from an RNA template. This process is the reverse of the usual flow of genetic information (DNA → RNA), hence the name reverse transcription.
Functions:-
> Converts viral RNA into DNA in retroviruses.
> The newly synthesized DNA integrates into the host cell's genome, allowing viral replication.
> Some reverse transcriptases also have RNase H activity, which degrades the RNA strand of an RNA-> DNA hybrid, and DNA polymerase activity to synthesize the second DNA strand.
Where it is found:-
> Retroviruses such as Human immunodeficiency virus (HIV).
> Retrotransposons (mobile genetic elements) in many organisms.
> Some DNA viruses (e.g., hepadnaviruses) also use reverse transcription during replication.
Applications in biotechnology:-
Reverse transcription PCR (RT-PCR):- Converts RNA into cDNA before amplification, widely used to detect RNA viruses and measure gene expression.
cDNA library construction:- Produces DNA copies of mRNA for cloning and sequencing.
RNA sequencing (RNA-seq):- Generates cDNA from RNA for transcriptome analysis.
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Lac operon in E. coli:-
· The operon controlling lactose metabolism. It consists of -
a) A regulatory or inhibitor (i) gene]:- It codes for the repressor.
b) 3 structural genes:-
i. z gene:- It codes for β-galactosidase which hydrolyze lactose to galactose and glucose.
ii. y gene:- It codes for permease which increase permeability of the cell to lactose.
iii. a gene:- It codes for transacetylase.
· The genes present in the operon function together in the same or related metabolic pathway. There is an operator region for each operon.
· In the absence of inducer:- If there is no lactose (inducer), Lac operon remains switched off. The regulator gene synthesizes mRNA to produce the repressor protein; this protein binds to the operator genes and blocks RNA polymerase movement. So the structural genes are not expressed.
· In the presence of inducer:- If lactose is provided in the growth medium, the lactose is transported into the E. coli cells by the action of permease. Lactose (inducer) binds with repressor protein. So repressor protein cannot bind to operator gene. The operator gene becomes free and induces the RNA polymerase to bind with promoter gene. Then transcription of structural genes starts. It is also known as indusible operon. Regulation of lac operon by repressor is called negative regulation.
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Transgenic plants: Application and achievements:-
Transgenic Plant:-
• Definition:- Stable transformation is achieved when a desired gene is integrated into the genome of a plant, this plant is called transgenic plant.
• These transgenic plants are developed for the following purposes:-
1. Insect Resistance
2. Virus Resistance
3. Seed Protein Quality
4. Gene Silencing
5. Male Sterility
6. Biochemical Production
1. Insect Resistance:-
cry gene transfer:- The cry gene is found in the plasmid of Bacillus thuringiensis (Bt) bacteria. This cry gene makes Crystal protein which is insecticidal.
Ø The cry gene was isolated from the plasmid of Bt and integrated into the cotton genome to form a transgenic plant called Bt-cotton.
Ø This Bt - cotton is resistant to boll worm.
Ø Crystal protein perforates the larva's alimentary canal leading to its death.
2. Virus Resistance:-
cp – gene transfer:- The coat protein is transferred from the TMV virus to the tobacco plant.
Ø The RNA of TMV virus is not come out in the presence of cp-protein. Due to which the tobacco plant becomes resistant to mosaic disease.
Ø Other examples: - Tomato, Alfalfa, Beetroot, Potato
3. Seed Protein Quality:-
SFA8 gene transfer:- In sunflower seeds, the SFA8 gene produces a protein that contains an excess of sulfur-rich amino acids methionine and cysteine. While the proteins present in pea seeds lack methionine and cysteine amino acids.
Ø Therefore the SFA8 gene is transferred to pea seeds.
4. Gene Silencing:-
Slow ripening tomatoes:-
Ø PG (Poly Galacturonase) enzyme digest the pectin. Due to which fruits become soft and fluffy quickly and ripen quickly.
Ø Anti-sense genes are integrated into the genome of tomato plants against genes that code PG enzymes. This inhibits the expression of PG gene and the tomato fruit ripens late.
Ø Antisense gene made against PG gene inhibits its expression, which causes tomato to ripen late and can be easily exported.
5. Male Sterility:-
Producing male sterility:-
Ø Flavonoid are essential for maturation of pollens. If we stop its formation then male sterility can be created in the plant.
Ø The CHS (Chalcone Synthase) enzyme is required for flavonoid synthesis.
Ø Anti-sense genes are integrated into the plant's genome against genes that code CHS.
Ø Absence of a flavonoid causes pollen to become inactive. The plant becomes male sterile.
6. Biochemical Production:-
• PHB (Poly Hydroxy Butyrate):- It is prepared from Acetyl CoA. From this bio-degradable plastic is made.
• The transgenic plant is developed by isolating the phb - B and phb - C genes from the Alcaligenes eutrophus bacterium and transfer to Arabidopsis thaliana. Now PHB is obtained from this plant.
Golden Rice:-
• The genes that form β - carotene are integrated into the embryo of rice within the genome to prepare golden rice.
• 2 genes are isolated from Daffodils plant and 1 gene from Erwinia uredovora bacterium.
• β - carotene is yellow in color, which makes rice yellow.
• β - carotene is the precurssor of vitamin A.
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Types of Hybrids:- There are 3 types of cellular hybrids -
1. Symmetric Hybrids
2. Asymmetric Hybrids
3. Cybrids
1. Symmetric Hybrids:- When the genomes of both species are kept in the cells during mitosis of the hybrid cell, the plants thus developed are called symmetric hybrids.
Example:- Pomato (Potato X Tomato)
2. Asymmetric Hybrids:- When the genome of a single species is extinct from cells during mitosis, the plants thus developed are called asymmetric hybrids.
Example:- Tobacco X Carrot
3. Cybrids:- When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.
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cDNA library:- It is a collection of cloned DNA sequences that are complementary to the mRNA that was extracted from an organism or tissue.
> cDNA is developed by the process of reverse transcription.
> Reverse transcription:- Genetic information contained in mRNA is converted back into a double stranded DNA form. The enzyme responsible for this is called reverse transcriptase.
> Reverse transcriptases:- They are isolated from Retroviruses. Eg.- Moloney murine leukemia virus (MMLV). They have polymerase activity, but do not have exonuclease activity.
> Primer:- MMLV will use mRNA as a template, but requires a primer. Eukaryotic mRNA has a 3' poly A tail. Therefore we can use poly dT as a prime.
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> RNAse H:- This enzyme has endonuclease activity, so create nicks in the RNA.
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> DNA polymese - I:- RNA fragments now serve as primers for DNA synthesis by E. coli Pol I. This enzyme also remove the RNA primers.
> T4 DNA ligase:- Nicks are sealed by this enzyme.
> Treat the cDNA with RNase H to remove possible 5' cap mRNA fragment remaining in cDNA duplex.
> Now treat the cDNA with S1 nuclease to remove 3' overhangs.
> Insertion of cDNA into plasmid:- 2 methods -
a. Homopolymeric tailing
b. Linker addition
a. Homopolymeric tailing:- Terminal transferase is an unusual DNA polymerase found only in prelymphocyte.
- In the presence of a divalent cation the enzyme catalyzes the addition of 3 to 1000 dNTPs to the 3'-OH end of DNA.
i. When the nucleotide to be added is a purine, Mg2+ is the cation used.
ii. When the nucleotide to be added is a pyrimidine, Co2+ is used.
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- Now treat the plasmid with PstI to ceate sticky ends and treat it with terminal transferase to add the complementary bases.
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- The utility of inserting the C-tailed cDNA insert into a G-tailed Pst I site in the vector is as follows:
i. The Pst I recognition sequence and cleavage site is:-
5' C T G C A ↓ G 3' →→→→→ 5' C T G C A 3' 5' G 3'
3' G ↑A C G T C 5' →→→→→ 3' G 5' 3' A C G T C 5'
ii. Cleavage of this site by Pst I, followed by G-tailing will produce:-
5' C T G C A 3' 5' G 3' →→→→→ 5' C T G C A (G)n 3' 5' G 3'
3' G 5' 3' A C G T C 5' →→→→→ 3' G 5' 3' (G)n A C G T C 5'
- Now anneal and ligate the cDNA into the plasmid.
b. Linker addition:-
- Linkers:- Linkers are short oligonucleotides (18 to 24 Ntds) which are typically palindromic and contain restriction endonuclease recognition sequence.
- If the ends of the cDNA fragments are blunt, then the linker can be ligated to both ends to introduce useful terminal restriction sites.
- The steps in linker addition are as follows:
i. Methylate cDNA at potential internal BamHI sites by treatment with BamHI methylase (plus S-adenosyl methionine).
ii. Ligate linkers to blunt, methylated cDNA using T4 DNA ligase.
iii. Cut linkers with BamHI restriction endonuclease.
iv. Remove linker fragments from cDNA fragments by agarose gel electrophoresis.
v.Ligate cDNA to vector DNA fragment (opened up by BamHI restriction endonuclease).
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Pollen Culture:-
1. Introduction:-
· Definition:- Pollen grain or microspore culture is an artificial process in which pollens or microspores are extracted from the intact anther at its uni-nucleated state and cultured on the nutrient medium under sterilized conditions.
· Androgenesis:- The process of artificial development of haploid plants by a series of cell division and differentiation from a totipotent pollen is called androgenesis. It is of two types -
i. Direct Androgenesis:- The microspore behaves like a zygote and by some changes the it forms a embryo like structure which further develops into haploid plants. This is called embryogenesis.
ii. Indirect Androgenesis:- The microspore divides repeatedly to form the callus tissue. Differentiation leads to development of haploid plant. This is called organogenesis.
2. Principle:-
· The haploid plant is developed using the totipotency of the microspore.
· Only one set of chromosomes is present in the microspore.
· In the process of haploid plant development, the normal development and function of the microspore of the male gametes formation stops. It is forced into a new metabolic pathway for somatic cell division.
· In pollen culture, diploid somatic cells of the anther also sometimes become active under culturing conditions and grow to form unwanted diploid callus or plantlets. Sometimes the chimera is formed. A callus or plantlet whose some cells are haploid and some cells are diploid, is called chimera. To avoid this problem, free pollens extracted from the anthers are cultured on the nutrient medium.
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Cosmids:-
> It is first described by Collins and Hohn in 1978.
> It is formed by joining ends of a linearized plasmid DNA with cos-site of lambda DNA.
> It is the commonly used cloning vector suitable for cloning large DNA fragments upto 45 kbp.
> Cosmid has an origin of replication, selectable markers, and gene cloning sites of plasmid DNA.
Salient features:-
i. Cosmid is a circular ds DNA.
ii. It has two complementary single-stranded regions at both ends of a plasmid DNA. The two cos-ends form a duplex by base pairing.
iii. The cosmid DNA does not code for phage proteins and host cell lysis.
iv. It does not involve in multiplication of phage particles.
v. It has an origin of replication from plasmid DNA for independent replication.
vi. It has selectable marker genes and gene cloning sites of plasmid DNA
vii. The cosmid DNA is packed within protein coat of bacteriophage to form inactive phage particles. Cos-site is a prerequsites for invitro packaging of cosmid in phage protein coat.
viii. After infection, the cosmid DNA does not integrate into host chromosomal DNA. It exits as a definite extra chromosomal DNA and replicates independently.
Examples:-
i. Cosmid pLFR5:-
- It is 6 kbp in size.
- It is constructed from E.coli plasmid pBR322 and two cos-ends of lambda DNA.
ii. Cosmid pJB8:-
- It is 5.4 kbp in size.
- It is constructed from the plasmid pBR322 and cos sites of lambda DNA.
iii. Cosmid pHC79:-
- It is 6.5 kbp in size.
- It is constructed from pBR322 and cos-sites of lambda DNA.
Advantages:-
- Cosmid pick up relatively larger DNA fragments than the plasmid do.
- As cosmids pick up large DNA fragments, they are used to establish gene libraries
- Gene cloning through cosmids helps in the study of non-sence sequences in the genome of organisms.
- Some cosmids are constructed by joining a linearized plasmid DNA with DNA fragments of p1
bacteriophage that have cos-ends. The P1 bacteriophage has the genome of 115 kbp. So, a DNA of 85
kbp can be packaged into the head of P1 phage. These cosmids help to clone large genes and gene
clusters in bacteria.
Disadvantages:-
- The packaging enzyme fails to pack recombinant cosmids into the phage head, if any one of the two
cos-ends is missing.
- Sometimes more than one recombinant cosmid join together to form a large DNA. If so, the packaging
enzyme fails to pack the DNA into the phage head.
- Slower replication
- Higher frequency of recombination inside bacterial host.
- Unstable inside E.coli host and thus easy to lose vector.
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