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What is a klenow fragment?

Klenow fragment: Definition and Applications

Definition: It is the large fragment of DNA polymerase I enzyme of E.coli produced by the proteolytic cleavage of DNA pol I by protease subtilisin.


klenow fragment Refer this figure for clear understanding. On the right side is the DNA Polymerase I before treating with protease. On the left side, upon treatment with protease, two fragments are formed, a large fragment called the klenow fragments and a small fragment with 5'→ 3' exonuclease activity.

This klenow fragment lacks 5'→ 3' exonuclease activity.

It has 5'→ 3' polymerase activity

It has 3'→ 5' exonuclease activity for proof reading activity

The other small fragment has 5'→ 3' exonuclease activity, but lacks 5'→ 3' polymerase activity and 3'→ 5' exonuclease activity of klenow fragment.


  • Synthesis of second strand on cDNA
  • DNA sequencing by the Sanger dideoxy method
  • Fill-in of 5´ overhangs to form blunt ends
  • Removal of 3´ overhangs to form blunt ends
  • Preparation of radioactive DNA probes.


Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

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Why Plasma membrane or Cell membrane is Semi-permeable or Selectively permeable?

Permeability of plasma membrane
Plasma membrane is a thin, semi-permeable membrane that serves as a boundary between the interior of a cell and its extracellular environment. It is present in all prokaryotic and eukaryotic cells.
it is also called as cell membrane, plasma lemma, bio membrane
In plant cells and bacterium, a thick outer wall is also present, called as cell wall.
Why Plasma membrane is selectively permeable or semi permeable?
Selective transport across plasma membrane
This property of selective permeability is responsible for keeping a cell ‘as a cell’, an individual self sustaining unit with a constant internal environment. Selectively permeable membranes can differentiate and discriminate between substances, allowing some to pass and others not.
It controls the entry of nutrients and exit of waste products from the cell. This trafficking allows cell to maintain an intracellular environment without the interference of outside substances. It takes in raw materials from the outside and synthesize complex organic molecules and keeping the products inside and remove waste products of these cellular activities.
The pH and volume of the cell is maintained by regulating the concentration of ions inside. The passive flow of ions into the cell without ATP loss or energy is achieved by creating a concentration gradient across the membrane by regulating ion concentration outside and inside of the cell.
What types of molecules that pass through the cell membrane?
The size of the molecules which can pass through the plasma membrane is 1-15 A0.
Gases like O2 and CO2 can diffuse rapidly in solution through membranes.
Small compounds like H2O and methane can easily pass through
Sugars, amino acids and charged ions are transported with the help of transport proteins.
The bulk transport of materials across the membrane takes place by exocytosis or endocytosis.
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How are Mitochondria and Chloroplast similar to Prokaryotic bacterial cells?

The similarity of mitochondrion and chloroplast with prokaryotic cells is the most compelling evidence that supports  Endosymbiont theory which explains the evolution of eukaryotic animal and plant cells. Refer figure.
Endosymbiont theory
Similarity between Mitochondrion and Prokaryotes
Similarity between prokaryotes and mitochondrion
In cell structure
  • The bacterial plasma membrane resembles the inner mitochondrial membrane in many aspects. In mitochondrion, the inner membrane is folded to form cristae which is the site of oxidative phosphorylation and electron transport and associated ATP production.
  • Bacterial plasma membrane has infoldings called mesosomes which is thought to be involved in respiration and DNA replication.
In Genetic material
  • Mitochondrion is a semi autonomous organelle with its own DNA called as mt DNA.
  • Like Bacterial DNA, mt DNA is circular, double stranded and not covered by a membrane.
  • Both Bacterial DNA and mt DNA  are devoid of histone proteins
  • Generally lack introns
  • Protein synthesis initiated by formyl methionyl tRNA (f-met tRNA) and in both mitochondria and bacteria protein synthesis can be inhibited by the same inhibitor chloramphenicol
  • Replication, transcription and translation similar to bacteria.
  • Sequence alignment studied reveal high degree of relatedness between mt genes and bacterial genes
In both cells, ribosomes are small 70 S ribosomes
Mitochrondria and chloroplasts replicate by a splitting process similar to binary fission in prokaryotes
Similarity between Chloroplast and Prokaryotes
Chloroplast is similar to mitochondrion in many aspects.
Similarity between bacteria and chloroplast
In Genetic material
  • Chloroplast is a semi autonomous organelle with its own DNA called as cp DNA.
  • Like Bacterial DNA, cp DNA is circular, double stranded and not covered by a membrane.
  • Both Bacterial DNA and cp DNA  are devoid of histone proteins
  • Generally bacterial DNA, mt DNA and cp DNA  lack introns
  • Small 70 S ribosomes.
  • Protein synthesis initiated by formyl methionyl tRNA (f-met tRNA).
  • Replication, transcription and translation similar to bacteria.
  • Transcription and translation are similar in chloroplasts and eubacteria: most chloroplast genes are transcribed as polycistronic units, their mRNAs are not capped, no poly(A) tails are added, and they possess a Shine Dalgarno ribosome binding sequence.
  • The RNA polymerase of liverworts contains α and β subunits and the amino acid sequence has great similarity to those of E.coli.
  • DNA sequences analysis suggest high degree of sequence similarity between cyanobacteria  and cp DNA of higher plants. Most cp DNA is thought to be evolved slowly in sequence and structure from cyanobacterial DNA.

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ICMR JRF 2015 Notification Apply Online Now

icmr jrf exam

ICMR in collaboration with PGIMER, Chandigarh will hold a National level examination for the award of Junior Research Fellowships (JRF), for Indian national candidates.
Exam Date: Sunday, July 19,2015.
A total of  120 fellowships awarded in Life Sciences stream (like microbiology, physiology, molecular biology, genetics, human biology, biotechnology, biochemistry, bioinformatics, biophysics, immunology, pharmacology, zoology, environmental sciences, botany, veterinary sciences. Another 100 candidates would be selected for consideration for positions of JRF under various research schemes of ICMR (subject to fulfilling the conditions for appointment under the schemes) for the duration of that scheme. These JRFs would also be permitted to complete Ph.D. while working in the scheme, if enrolled.  The validity of result will be two years for placement in ICMR funded projects.

Important Dates

  • Date of opening of Registration: 13.04.2015
  • Last date for filling of online application: 12.05.2015
  • Late date of fee deposit in Bank: 15.05.2015
  • Date of Entrance Examination (Tentative): 19.07.2015
Duration and Emoluments: The existing value of the fellowship is at present Rs. 25000/- (Rupees Twenty Five  thousand only)  Per  month and an annual contingency grant up to Rs. 20,000/- per annum. The local institution shall review the performance of JRF after two years through an appropriate Review Committee constiuted by the Head of the institution. The fellow may be awarded SRF after successful assessment by the Review Committee.
Age Limit: The age limit for admission to the eligibility test is 28 years (upper age limit relaxable upto 5 years in case of candidates belonging to SC/ST, physically handicapped (PH) and female candidates, 3 years in the case of OBC category.
Examination Centre :  01) Chandigarh, 02) Chennai, 03) Delhi 04) Kolkata, 05) Mumbai, 06) Hyderabad  07) Guwahati 08) Varanasi 09 Bhopal Code Name of the Centre 10) Bhubaneshwar 11) Sri Nagar 12) Bangaluru
Syllabus: As prescribed by UGC.
Scheme of Test
The test will consist of one paper of 2 hours duration. The paper will consist of 2 Sections.
The Aptitude Section (Section A) will have 50 questions on
(i) scientific phenomenon in everyday life;
(ii) general knowledge in sciences; and
(iii) common statistics.
All these questions would be compulsory with each question carrying 1 mark. The subject Specific Section (Section B & C) would pertain to (B) Life Sciences and (C) Social Science. The candidate may attempt questions in either of the two areas. Each area of section B & C would have 100 questions and the candidate may attempt any 75 questions in the predesigned area of Section B or C.
Candidates are required to indicate the option for Section B or C in the application form too.
Each question carries one mark. Negative marking @ 0.25 will be made for each of
the wrong answer
. The questions in both the sections would appear in English only.
The qualifying marks will be 55% obtained in both the sections (A+B or C) for General
Category and OBC and 50% for SC/ST and physically handicapped.
Subjects covered under Life Sciences include microbiology, physiology, molecular biology, genetics, human biology, biotechnology, biochemistry, bioinformatics, biophysics, immunology, pharmacology, zoology, botany, environmental sciences and veterinary science.

  • Free ICMR JRF Preparation Resources
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    Enzymes in rDNA or Recombinant DNA Technology

    The major discovery that led to the birth of rDNA technology was the discovery of restriction enzymes or enzymes to cut DNA molecule.
    Previously, we have discussed in detail the steps in rDNA technology. Let us start with definition.
    What is recombinant DNA or rDNA technology?
    It is the sum of techniques used in genetic engineering that involves the identification, isolation and insertion of gene of interest into a vector such as a plasmid or bacteriophage to form a recombinant DNA molecule and production of large quantities of that gene fragment or product encoded by that gene.
    Many enzymes are used in rDNA technology. Let us have a quick glance of the enzymes used in rDNA technology.
    Enzymes in rDNA technology or recombinant DNA technology
    I. Nucleases: Enzymes to hydrolyse nucleic acids, DNA or RNA. Restriction Enzymes (called as Molecular scissors, Molecular scalpels etc)
    a) Restriction endonuclease: Makes cuts at specific sites within the DNA Eg: Eco R1. Restriction site of Eco R1 is GAATTC.
    b) Restriction exonuclease: Remove nucleotides from the ends.
    c) Ribonuclease-H (RNase-H):
    Ribonuclease H or Rnase H mechanism
    Selectively removes the mRNA from RNA DNA hybrid to separate the cDNA to synthesize the second strand.
    It is isolated from retroviruses such as AMV, MMTV etc.
    II. DNA Modifiers: Enzymes involved in modification of DNA strands to make it suitable for rDNA technology
    a) DNA polymerase: It is involved in elongation of the strand. It is a template directed enzyme that adds complementary nucleotides to the free 3’ OH end of the strand. Commonly used DNA polymerase is Taq DNA polymerase isolated from Gram negative bacterium Thermus aquaticus
    DNA polymerase in rDNA technology
    Uses: in Polymerase Chain reaction. In DNA sequencing by Maxam-Gilberst method. In making DNA probes for DNA finger printing.
    b) Reverse Transcriptase: Enzyme that makes DNA strand using mRNA as template strand.
    Reverse transcriptase in rDNA technology
    Also called as RNA dependant DNA synthetase. Used in the making of cDNA from mRNA for cDNA library preparation.
    c) Alkaline Phosphatase :This enzyme removes terminal phosphate group (PO4-2 ) at the 5’ end of a DNA or RNA.
    Alkaline phosphatase in rDNA technology
    Uses: Removal of terminal phosphate group at the 5’ end of a DNA prevents self annealing of vector DNA .
    In radioactive labeling using P32, phosphate group at the 5’ end is removed and replaced with P32 using alkaline phosphatase.
    d) Polynucleotide kinase: It transfers or add phosphate from ATP to 5’OH group of dephosphorylated DNA or RNA.
    Polynucleotide kinase in rDNA technology
    Uses: To rephosphorylate the 5’ end of dephosphorylated DNA (vector DNA) or RNA.
    Used in radiolabelling: to transfers radioactive P32 from ATP to dephosphorylated 5’ end of DNA or RNA.
    e) Terminal nucleotidyl transferse : Enzyme that adds nucleotides to 3’OH group of a DNA fragment.
    Terminal nucleotydyl transferase in rDNA technology
    Uses: used to make homopolymer sticky or cohesive tails at 3’ end of DNA fragment. This helps in joining fragments with blunt ends.
    Used to make radioactive DNA probes by end labeling.
    f) Methyl transferase : Methyl Transferase: enzyme that adds a methyl group to cystine and adenine of DNA
    Methyl transferase in rDNA technology
    Uses: To Methylate desired DNA in the rDNA to protect it from cleavage by restriction enzymes of restriction modification systems of the host. To protect restriction sites from a restriction enzyme , if the target DNA has many sites fro that particular restriction enzyme.
    III.Ligases: ‘joining enzyme’
    Joining enzyme that joins the ends of 2 double stranded DNA molecule. The process is called ligation.
    The bond formed is called phosphodiester bond.
    Ligase in rDNA technology
    Between the 5’P of a nucleotide of one DNA fragment and the 3’ OH end of the other. Requires ATP and NAD+ for its activity. Eg: T4 ligase
    Try it now Multiple choice Quiz on Enzymes in rDNA technology
    Learn more Definition and Steps in rDNA tecchnology
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    ABC Model of Flower Development in Arabidopsis thaliana

    Flower is considered as a modified shoot. Many genes are involved in the conversion of shoot meristem to floral meristem.
    ABC model of floral development explains the genetics behind the formation of different whorls in a flower.
    The four whorls are sepal, petal, stamens and carpel.
    ABC model whorls
    Flower formation is initiated by the activity of genes known as heterochrony or flowering time genes which regulate the conversion of vegetative meristem to floral meristem. Then flower meristem identity genes regulate the formation of flower. Upon initiation of flowering, a third type of genes called cadastral genes will express which govern the whorl formation of the flower. Finally the structure of this whorls and its occurrence at right place is regulated by homeotic genes.
    The four whorls are sepal, petal, stamens and carpel.
    ABC model was first formulated by George Haughn and Chris Somerville in 1988. Experimental material was Arabidopsis thaliana and Antirrhinum majus.
    ABC model predicts that the four whorls of flower are controlled by the action of 3 genes A, B and C in Arabidopsis thaliana. The role of these genes was deduced by inducing mutation in each of these genes.
    Key 1: Remember, A and C genes are equally dominant. Mutation of ‘A’ gene makes ‘C’ gene more active.
    Key 2: B genes always express in association with A and C.
    ABC Model of flower development summary chart
    The above figure summarizes the ABC model and changes associated with whorl formation on mutation of A, B and C genes respectively.
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    DBT BET JRF Exam 2015 Notification

    Biotech Students Don’t miss this exam!
    Applications are invited from Indian nationals for the award of “DBT-Junior Research Fellowship” (DBT-JRF) for pursuing research in frontier areas of Biotechnology and Applied Biology.
    JRFs will be selected according to merit under two categories: Category I & II.
    dbt bet jrf exam
    Category I fellowship (Top 275 in number) are tenable in any University/Institute in India where the students can register for Ph.D.
    Category II students (100 in number) will be eligible to join any DBT sponsored Project and avail fellowship equivalent to NET/GATE qualifications as per DST Guidelines, subject to selection through institutional selection process. Fellowship will be co-terminus with the duration of project and institutional rules will be applicable. There will be no binding on PIs of DBT sponsored projects to select JRF/SRF for their projects from category II list. Selection in Category II will not entitle student for any fellowship from DBT-JRF program.
    For further details please visit .
    Students can also visit NCCS website for details:
    Exam Date: 26 th April, 2015 at 2.30 PM  (Online Test)
    Eligibility: Candidates can appear for BET examination only thrice during their career within the prescribed age limit. Candidates who have completed eligibility qualification in a year will be eligible to appear in that year and two subsequent years. Students who have passed after Jan.1, 2013 or will appear this year (till August, 2015) final examination of qualifying degree is eligible. Students with M.Sc. / M.Tech / M.V.Sc. degree with Biotechnology in title of degree e.g. Biotechnology, specialization such as Agricultural, Animal / Veterinary, Medical, Marine, Industrial, Environmental, Pharmaceutical, Food, Bio-resources Biotechnology, “Biochemical Engineering, Bio-sciences and Biotechnology, Bioinformatics” and M.Sc. “Molecular & Human Genetics” and M.Sc. “Neuroscience” as well as B.Tech / B.E. in Biotechnology (4-year course after 10+2) recognized by UGC/AICTE are eligible for this examination.
    The applicants should be below the age of 28 years for Open category as on 28th February 2015.
    Candidates with minimum 60% for general and OBC category (55% for SC/ST/PH) of the total marks (equivalent grade) are only eligible.
    For further details and to apply online please visit the URL . Online registration will start on 25 th February 2015 and the last date for submission of online application form is 25 th March, 2015. DBT-BET (category I) entitles a candidate for fellowship subject to Ph.D registration of the candidate in a recognized university or Institute in the country within 2 years. At present, very few institutes or universities allow B.Tech students to register for Ph.D. directly. Registration for Ph.D is candidate’s responsibility and NCCS or DBT have no role in this.
    Remember: For lectureship eligibility still you have to qualify NET.
    Reservations: Government of India guidelines for reservations will be followed for BET-2015. Age relaxation of 5 years (up to 33 years) for SC /ST/PH and women candidates and 3 years (31 years) for OBC candidates will be given. The age limit will be as on 28th Feb. 2015. SC/ST/PH candidates with 55% of the total marks (equivalent in grade) are eligible to apply.

    Fellowship: The fellowship will be initially for a period of 3 years extendable for 2 more years based on performance. By the end of 2nd year, the performance of JRF will be assessed and will be upgraded to SRF. The fellowship for JRF/SRF will be @ Rs. 25,000/- or 28,000/- per month + HRA as per DST guidelines and research contingency of Rs. 30,000/- per year.

    Mode of selection: The candidates will be selected based on an online admission test, “Biotechnology Eligibility Test” (BET) to be conducted on 26 th April, 2015 at 2.30 PM in the following twelve cities: New Delhi, Kolkata, Guwahati, Hyderabad, Chennai, Pune, Bangalore, Chandigarh, Lucknow, Patna, Trivandrum & Ahmedabad.

    Mode of application: Candidates should register and apply online in the prescribed application form available at the URL starting on 25 th February 2015. All the details about the application process and the examination are available on the web site. The last date for submission of online application form is 25 th March, 2015. Stepwise procedure for filling the online application form, payment of application fees and uploading of required documents/certificates is given in the above said URL. General/OBC candidates have to pay an application fee of Rs. 500/- and SC/ST/PH categories are exempted from payment of application fees. The application fees is payable either online or offline as detailed on the web site for completion of application process. The application fee is non-refundable and non-transferable. No TA will be provided to any candidate for attending the examination.
    Scanned Copies of following Documents will be required for filling up the application form online
    Specification for scanning the documents is available on website.
    1. Passport size photo
    2. Signature
    3. Date of birth proof (preferably SSC certificate)
    4. Final mark sheet of the qualifying examination showing the cumulative percentage of marks or equivalent CGPA. 5. For students appearing in the final examination in 2015, filled and signed certificate in the format available at website.
    6. If the candidate belongs to SC/ST/PH/OBC category, they should scan suitable certificate issued by the competent authorities. OBC candidates should produce ‘Non-creamy layer certificate’ for availing the relaxation.
    Free DBT-BET-JRF Exam Preparation Resources
    First and foremost thing is to begin the preparation now onwards
    " Wishing the very Best "
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    B cell-T helper cell interaction and associated antibody production: Step wise explanation

    Humoral immunity: Pathway 2 B cell as antigen presenting cell (APC)
    Definition: Humoral immune response refers to the host defense mechanisms that are mediated by antibodies produced by plasma cells of B cells.
    It protects against extracellular bacteria and foreign macromolecules.
    There are two pathways in antibody production
    Pathway 1: B Cell Mediated Pathway which is independent of T helper cells.
    We have already discussed this pathway in the last post.
    Pathway 2: B Cell- T helper Cell Mediated Pathway
    In this pathway, B cell acts as antigen presenting cell

    B cell-T helper cell mediated antibody production
    Step 1:
    B cell activation=Antigen binding to Naïve B cell receptor.
    Antigen degradation inside B cell
    B cell possesses B cell receptor or BCR with a single specificity. A naive B cell is the one which hasn't encountered an antigen before. When an epitope of an antigen binds to the B cell receptor, that particular B cell gets activated.
    Step 2:
    B cell as antigen presenting cells (APCs)
    In this pathway, B cells acts as antigen presenting cell that is presenting antigen to T helper cells.
    MHC class II receptor is present on all antigen presenting cells like B cells, macrophages, dendritic cells etc.
    The antigen is processed inside B cell and is presented on MHC class II to T cell receptor of T helper cell. This MHC class II bound antigenic peptide binds to TCR. T helper cells secrete chemokines or chemical messengers which in turn activates B cells more effectively. T helper cell mediated B cell activation is more effective than independent B cell activation.

    Step 3:
    Clonal selection and Differentiation:
    Division of that activated B cell
    This activated B cell is selected to divide forming many copies of that cell. That particular clone of B cell is selected to divide and is called as clonal selection.
    Differentiation: Plasma cells and memory B cells
    Later this B cells differentiate to form plasma cells or effecter cells and memory B cells
    Step 4:
    Plasma cells produce antibodies that bind to the antigen and ensure its clearance from the system by agglutination, precipitation or neutralization.
    Memory B cells are meant for immunologic memory or secondary immune response. When the same antigen comes for the second time, these memory B cells will recognize it quickly and induce a heightened immune response. This will clear out the pathogen from the system soon.

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