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PCR short notes - Definition, Requirements, Steps and Applications

PCR: Polymerase Chain Reaction
• Invented by Kary Mullis 1983
• Received Nobel Prize in chemistry in 1993
Definition: An in-vitro DNA amplification technique that allows synthesizing millions of copies of the gene or DNA of interest from a single copy
• It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.



Requirements or Reaction components:
1. Target DNA - contains the sequence to be amplified.
2. Two oligonucleotide primers : binds to the 3’ OH end of both strands
3. dNTPs – deoxy nucleotide triphosphates: DNA building units.
4. Thermostable DNA polymerase (Taq polymerase)-enzyme that catalyse the reaction
5. Mg++ ions - cofactor of the polymerase enzyme.
6. Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme.
  1. PCR steps
Steps in PCR
A PCR cycle consist of 3 steps: Denaturation, Primer annealing and Elongation.
Step I: Denaturation
• Reaction mixture heated to 940C for 1 minute.
• The temperature denatures dsDNA into two single strands by breaking H-bond between them
Step 2: primer annealing
• Cooled to 550C for 1.5min
• Primer binds to the 3’ end of the target DNA at both strands-primer annealing
Step 3: Elongation by thermostable DNA polymerase
• At 720 C for 1 min
• Addition of nucleotides by Taq polymerase
• Duration to complete a cycle ~4-5 min
• In each cycle the no. of DNAs gets doubled-2->4->8->16….exponentially
• A reaction usually consist of 30-35 cycle forming about million copies
Applications of PCR :New applications are created every day,
• To amplify DNA fragments isolated from organisms
• To propagate DNA for gene manipulation and construction of DNA libraries
• Used in sex determination of embryo
• In forensic science, in DNA fingerprinting
• PCR products can be used for mapping genes
• PCR products can be used as probes
• Used to detect genetic diseases
• PCR can be used to identify genotypes
• PCR can be used to sequence DNA directly.
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ICMR JRF 2014 Notification, Eligibility, Previous Questions

ICMR in collaboration with PGIMER, Chandigarh will hold a National level examination for the award of Junior Research Fellowships (JRF), for Indian national candidates.
ICMR JRF EXAM
Examination Centre Code:  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
Exam Date: Sunday, July 13,2014.
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 ).
Eligibility:
Educational: M.Sc/M.A or equivalent degree with minimum 55% marks for General/OBC Candidates and 50% for the SC/ST & physically handicapped (PH) candidates in the subjects mentioned above.
Candidates appearing in the final year examination (2014-2015) can also apply.
Age Limit: The age limit for admission to the eligibility test is 28 years as on 30-09-2014 (upper age limit relaxable up to 5 years in case of candidates belonging to SC/ST, physically handicapped (PH) and female candidates, 3 years in the case of OBC category.
Important Dates to remember
  • Last Date for receipt of application 30.04.2014
  • Late Date for receipt of application 5.05.2014
  • For Remote Areas Date of Entrance Examination 13.07.2014
Syllabus: • As prescribed by UGC.
• A dummy sample question paper for ICMR JRFs Examination is available on
PGIMER/ICMR website
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.
How to apply
The candidate is required to download the Application Form/Challan Form from the PGIMER
Website http://pgimer.edu.in and ICMR New Delhi website http://icmr.nic.in only. After filling the application form the candidate is required to send it by Speed Post/Registered Posts to Registrar, PGI, Chandigarh on or before 30.4.2014. After depositing the fee with the branch of State Bank of India, the candidate shall send PGI’s copy of the Challan Form along with the application form and retain candidate’s copy of Challan with him/her for future use. The third copy of the challan form will be retained by the State Bank of India. The Challan form is required to be filled up in the name of Director, PGIMER, Chandigarh in the ‘Power Jyoti’ Account No. 32211616762. In that case the INB reference No. will have to be mentioned instead of Challan No. in the application form. The candidate is required to send two window envelopes (without self-address) affixing Rs. 10/- postal stamp on them directly to Registrar, PGI, Chandigarh before 30.4.2014.
Amount of Application Fee & Mode of Payment:
The candidates can go to any branch of State Bank of India with the fee payment challan duly filled in and pay the prescribed application fee in the “Power Jyoti” PUL current account of ICMR. The Account No. of ICMR for admission is 32211616762. The candidates after having deposited the fee in the bank must ensure that they have Triplicate Fee Payment Challan with Challan No. given by the bank on it. The candidate should keep one copy of this Challan Form with them for future use.
The amount of fee to be paid is as under :
General & OBC : Rs. 500/-
SC/ST/OPH : Rs. 300/-
No other form of payment of application fees like Demand Draft, Banker Cheque, Money Order,
Postal Order etc. will be accepted under any circumstances. The fee once paid will not be
refunded.


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    Difference between Prokaryotic and Eukaryotic Gene Expression

    Gene expression is the mechanism at the molecular level by which agene is able to express itself in the phenotype of an organism.  The mechanism of gene expression involves biochemical genetics. It consists of synthesis of specific RNAs, (i.e., transcription) polypeptides, structural proteins, proteinous biochemicals or enzymes (i.e., translation)  which control the structure or functioning of specific traits.
    Gene expression in eukaryotes is much more complex than in prokaryotes. The differences are follows:
     prokaryotic gene expression
    Prokaryotic Gene Expression:
    1. Transcription Enzyme: One RNA polymerase for all RNA’s
    2. Enhancer: Transcription enhancers are not required.
    3. Primary transcript: Directly function as mRNA.
    4. mRNA: Polycistronic
    5. Location of transcription: Cytoplasm (Nucleoid region)
    6. Coupling of transcription and translation: Occurs
    7. Translation initiation factors: Three represented as IF-1, IF-2, IF-3
    8. Exit of deactylated tRNA: From E site of ribosomes
     Eukaryotic gene expression
    Eukaryotic Gene Expression:
    1. Transcription Enzyme: At least three (RNA polymerase I for rRNA, RNA polymerase II for hnRNA, RNA polymerase III for tRNA, 5SRNA  and snRNA)
    2. Enhancer: Alongwith promoter region, enhancers are also required.
    3. Primary transcript: It is hnRNA that contains both introns and exons. Processing removes introns and produces functional mRNA.
    4. mRNA: Monocistronic
    5. Location of transcription: Nucleus
    6. Coupling of transcription and translation:
    7. Translation initiation factors: Nine represented as eIF where e donates eukaryotes. These are eIF2, eIF3, eIFIA, eIF4B, eIF4E, eIF4G, eIF5 & eIF6.
    8. Exit of deactylated tRNA: From P site as E site is absent in eukaryotic ribosomes.
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    Bonds used in Protein Structure

    Proteins are the most abundant biological macromolecules occurring in all cells and all parts of cells. These are the molecular instruments through which genetic information is expressed. All proteins are constructed from the same ubiquitous set of 20 amino acids, covalently linked by peptide bonds in characteristics linear sequences. Amino acids condense to produce peptides. the bond formed is called peptide bond.
    Bond used in Protein Structure
    1. Peptide bond: It is formed when a water  molecule is eliminated during a reaction between –NH2 of one amino acid and –COOH of another.
    peptide bond
    2. Ionic bond occurs  due to attractive force between oppositely charged ionised groups.

    3. Disulphide bond may be formed between different chains of amino acids or between different parts of the chain. It provide some rigidity to the protein molecule.
    Bond used in Protein Structure
    4. Hydrogen bond develops due to sharing of H+ by two eletronegative atoms. It is a weak bond.

    5. Hydrophobic bond is formed between two non polar groups. It help in excluding water in that area and increasing compaction.
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    DBT BET JRF 2013 Questions and Answers

    111. SNARE proteins are found in the membranes of all of the following compartments EXCEPT
    a) Mitochondria
    b) Golgi complex
    c) Early endosome
    d) Endoplasmic reticulum

    112. All of the following enzymes are linked to reduction of NADH except
    a) isocitrate dehydrogenase
    b) lactate dehydrogenase
    c) succinate dehydrogenase
    d) pyruvate dehydrogenase

    113. Which of the following are components of a phospholipid?
    a) cholesterol, glycerol, fatty acids
    b) fatty acids, phosphate group, glycerol
    c) glycerol, amino acids, phosphate group
    d) phosphate group, cholesterol, monosaccharides

    114. Prosthetic groups such as iron-sulfur clusters and heme function to
    a) Donate electrons to NADH
    b) Allow proteins to diffuse within the mitochondrial inner membrane
    c) Both accept and donate electrons during electron transport
    d) Transport protons across the mitochondrial inner membrane

    115. Sciophytes are plants that prefer to grow in
    a) Sun
    b) Shade
    c) Cold temperature
    d) Water

    116. Which one of the following is a micronutrient of plants?
    a) Mn
    b) P
    c) Ca
    d) Mg

    117. Which of the following statements about the trp operon is not correct?
    Trp operon
    a) It is normally turned off if tryptophan is present.
    b) Tryptophan acts as the corepressor.
    c) The regulator gene product is inactive by itself.
    d) Tryptophan binds to the repressor protein and inactivates it.

    118. Glucose labeled with 14C at C-6 is added to a solution containing the enzymes and cofactors of the oxidative phase of the pentose phosphate pathway. The radioactive label will be observed at
    a) C5 of ribulose 5-phosphate
    b) C3 of ribulose 5-phosphate
    c) C1 of ribulose 5-phosphate
    d) C4 of ribulose 5-phosphate

    119. The molarity of 15 % NaCl is
    a) 2.56
    b) 0.256
    c) 25.6
    d) 0.025

    120. Which one of the following antibiotics is used to demonstrate the fresh protein synthesis in response to challenge by an inducer?
    a) Chloramphenicol
    b) Carbenicillin
    c) Rifampicin
    d) Tetracyclin
    Learn more:
    Answers
    111. a) Mitochondria
    112. c) succinate dehydrogenase
    113. b) fatty acids, phosphate group, glycerol
    114. c) Both accept and donate electrons during electron transport
    115. b) Shade
    116. a) Mn
    117. d) Tryptophan binds to the repressor protein and inactivates it.
    118. a) C5 of ribulose 5-phosphate
    119. a) 2.56 (Molarity = No of moles of solute/Litre of Solution)
    120. a) Chloramphenicol
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    DBT BET JRF Exam Questions 2013

    101. Mammalian cells in primary culture experience Heyflick limit after 50-60 cell generation time. However, in a rare situation or employing certain agents, cells can be induced to immortality. Which of the following genes/proteins have a major relationship with this?
    a) Cdc2
    b) Cyclin C
    c) p53
    d) Proteasomes

    102. In the preparation of Golden rice, a few exogenous genes have been used in order to achieve the production of Vitamin A through recombinant DNA technology. This is a true example of
    a) metabolic repression
    b) biochemical engineering
    c) metabolic extension
    d) combinatorial biosynthesis

    103. Which one of the following is a signaling receptor?
    a) mannose receptor
    b) toll-like receptor
    c) scavenger receptor
    d) LPS receptor

    104. While sedimenting a microparticle by centrifugation, instead of increasing the rpm (to attain the desired “g” force), what else can be done to attain the complete sedimentation?
    a) Decrease the density of the medium
    b) Increasing the density of the medium
    c) By changing the Ph
    d) Increasing the sample volume

    105. For passive vaccination, which antibody type will be most appropriate?
    a) Polyclonal antibody
    b) Monoclonal antibody
    c) Humanized antibody
    d) Single chain antibody

    106. Which of the following is the best method to determine the phospholipid asymmetry in a plasma membrane?
    a) electron microscopy
    b) fluorescence spectroscopy
    c) lectin binding
    d) Thin layer chromatography

    107. Amino acid analysis of 1.0 mg of pure enzyme yielded 58.1 µg of leucine (MW = 131.2). What is the minimum MW of the enzyme based on leucine content?
    a) 2000
    b) 2300
    c) 2350
    d) 2258

    108. The first metabolic intermediate that is common to the aerobic metabolism of glucose and fatty acids is
    a) acetyl CoA
    b) aceto-acetyl CoA
    c) Pyruvate
    d) Citrate

    109. APS (Ammonium persulphate) is used in SDS-PAGE for
    ammonium persulfate function in sds-page
    a) preventing oxidation
    b) cross-linking
    c) its role as a catalyst
    d) free radical formation

    110. Identify the mismatch
    a) Alkaline phosphatase : remove phosphate group present at 5’ end of DNA
    b) DNA Polymerase I : nick translation
    c) S1 endonucleases : cleaves only single strand DNA
    d) DNase I : cleaves only double stranded DNA
    Learn more:
    Answers
    101. c) p53 (Read more: Apoptosis and genomic instability)
    102. c) metabolic extension
    103. b) toll-like receptor
    104. a) Decrease the density of the medium
    105. a) Polyclonal antibody
    106. d) Thin layer chromatography
    107. d) 2258
    108. a) acetyl CoA
    109. d) free radical formation (Polymerization of acrylamide and bisacrylamide monomers is induced by APS (ammonium per sulphate ), which spontaneously decomposes to form free radicals)
    110. d) DNase I : cleaves only double stranded DNA (It acts on single-stranded DNA, double-stranded DNA, and chromatin.)
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