Tag: NEET PG

Central Dogma Of Molecular Biology

CENTRAL DOGMA OF MOLECULAR BIOLOGY


Central  Dogma  Of  Molecular Biology 

  • The flow of information from DNA to RNA to protein is ter med the “central dogma” of molecular biology.
  • It is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information.
  • DNA stores genetic information:- Information about amino acid sequence of all the proteins is present in the form of genesin DNA. The entire genetic material present in the DNA of an organism is known as genome.The important role of DNA in transfer of information in living cells is called central dogma of molecular biology.
  • According to the central dogma, information flows from DNA to RNA to protein. This occurs in three step:

                   1) DNA replication.

                   2) Transcription

                   3) TransIation

  •  In DNA replication there is copying of parent DNA to form daughter DNA molecules having nucleotide sequence indentical to those of the parent DNA.
  • In Transcription there is the process in which the genetic messages in DNA are rewritten in the form of RNA. In simple words, Synthesis of RNA from DNA is called transtiption (flow of information lromDNA to RNA).
  • In translation, the genetic message coded by RNA is translated by the ribosomes into the protein structure, i.e.synthesis of proteinfrom RNA.
  • Biosynthesis of protein is under direct control of DNA in most cases or else under the control of genetic RNA where DNA is absent. Sequences of bases in a particular segment of a polynucleotide chain will determine the sequence of amino acids in a particular polypeptide.
  • The relationship is popularly known as central dogma. Flow of information is one way i.e., from DNA, information is transferred to RNA (mRNA) and from RNA to protein.
  • Temin (1970) reported that retroviruses operate a central dogma reverse or teminism inside host cells. Genomic RNA of these viruses first synthesizes DNA through reverse transcription. DNA then transfers information to messenger RNA which takes part in translation of the coded information to form polypeptide.

1) One way flow of information (central dogma)

DNA(transcription) → RNA (translation) → proteins

(2) Reverse flow of transcription information

DNA(transcription) ↔RNA(translation) → proteins

Exam Important
  • In translation, the genetic message coded by RNA is translated by the ribosomes into the protein structure, i.e.synthesis of proteinfrom RNA.
  • Biosynthesis of protein is under direct control of DNA in most cases or else under the control of genetic RNA where DNA is absent. Sequences of bases in a particular segment of a polynucleotide chain will determine the sequence of amino acids in a particular polypeptide.
  • The relationship is popularly known as central dogma. Flow of information is one way i.e., from DNA, information is transferred to RNA (mRNA) and from RNA to protein.
  • Temin (1970) reported that retroviruses operate a central dogma reverse or teminism inside host cells. Genomic RNA of these viruses first synthesizes DNA through reverse transcription. DNA then transfers information to messenger RNA which takes part in translation of the coded information to form polypeptide.

(1) One way flow of information (central dogma)

DNA(transcription) → RNA (translation) → proteins

(2) Reverse flow of transcription information

DNA(transcription) ↔RNA(translation) → proteins

Don’t Forget to Solve all the previous Year Question asked on CENTRAL DOGMA OF MOLECULAR BIOLOGY

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Central Dogma Of Molecular Biology

CENTRAL DOGMA OF MOLECULAR BIOLOGY

Q. 1 Assertion : Central dogma is the flow of information from DNA to mRNA and then decoding the information present in mRNA in the form of protein.
Reason : In retroviruses, reverse of central dogma occurs.

 A

If both the assertion and the reason are true and the reason is a correct explanation of the assertion

 B

If both assertion and reason are true but the reason is not the correct explanation of the assertion

 C

If the assertion is true but the reason is false

 D

If both the assertion and reason are false

Q. 1

Assertion : Central dogma is the flow of information from DNA to mRNA and then decoding the information present in mRNA in the form of protein.
Reason : In retroviruses, reverse of central dogma occurs.

 A

If both the assertion and the reason are true and the reason is a correct explanation of the assertion

 B

If both assertion and reason are true but the reason is not the correct explanation of the assertion

 C

If the assertion is true but the reason is false

 D

If both the assertion and reason are false

Ans. B

Explanation:

Answer: B If both assertion and reason are true but the reason is not the correct explanation of the assertion

  • Biosynthesis of protein is under direct control of DNA in most cases or else under the control of genetic RNA where DNA is absent. Sequences of bases in a particular segment of a polynucleotide chain will determine the sequence of amino acids in a particular polypeptide.
  • The relationship is popularly known as central dogma. Flow of information is one way i.e., from DNA, information is transferred to RNA (mRNA) and from RNA to protein.
  •  Temin (1970) reported that retroviruses operate a central dogma reverse or teminism inside host cells. Genomic RNA of these viruses first synthesizes DNA through reverse transcription. DNA then transfers information to messenger RNA which takes part in translation of the coded information to form polypeptide.

proteins

Quiz In Between



Molecular Cytogenetic Technique- (Fish)

MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)


  • FISH is a cytogenetic technique that can be used to detect the presence or absence ofspecific DNA sequences (specific gene locus).
  • fluorescent probes bind to only those parts of the chromosome with which they show a high degree of sequence similarity.like lncRNA, mRNA, or miRNA of interest
  • FISH  can  be used for  rapid  identification  of chromosome during  interphase.
  • FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine qtogenetics or identify extra material of unknown origin.
  • it determines if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
  • This technique can be used on formalin-fixed paraffin embedded (FFPE) tissue, frozen tissues, fresh tissues, cells and circulating tumor cells.

Methodology:

  • (FISH) analysis is performed by denaturing the double-stranded DNA in the fixed chromosomes on a microscope slide.
  • Two fluorescently labeled DNA probes are used in combination to analyze each location.
  • The first probe serves as a control and hybridizes with DNA on the target chromosome, The second probe hybridizes to a target location on the individual’s DNA sequence.
  • When a deletion is present, the second probe will not hybridize and no fluorescence will be seen.
  • A duplication will result in two fluorescent spots with the test probe.

Advantages of FISH

  • FISH permits determination of the number and location of specific DNA sequences in human cells.
  • FISH can be performed on metaphase chromosomes,as with G-banding, but can also be performed on cells not actively progressing through mitosis
  • FISH performed on nondividing cells is referred to as interphase or nuclear FISH.

Disadvantages of FISH

  • FISH requires a preselection of an informative molecular probe prior to analysis, So a prior knowledge of the anomaly is needed.

Uses  of  FISH:

  • Detection of numeric abnormalities of chromosomes (aneuploidy). The demonstration of subtle microdeletions
  • Detection of complex translocations not detectable by routine karyotyping
  • For analysis of gene amplification e.g. HER2/NEU in breast cancer or N-MYC amplification in neuroblastomas
  • For mapping newly isolated genesa of interest to their chromosomal loci

Chromosome  Painting:

  • Is an extension of FISH, Probes are prepared for entire chromosomes.
  • The number of chromosomes that can be detected simultaneouslyby chromosome painting is limited by the availability of fluorescent dyes.

Exam Important

  • FISH  can  be used for  rapid  identification  of chromosome during  interphase.
  • FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine qtogenetics or identify extra material of unknown origin.
  • it determines if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
  • It helps in Detection of numeric abnormalities of chromosomes (aneuploidy). The demonstration of subtle microdeletions

 

Don’t Forget to Solve all the previous Year Question asked on MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)

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Molecular Cytogenetic Technique- (Fish)

MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)

Q. 1 Which of these is not a method for introducing genes into a cell?

 A

Transfection

 B

Electroporation

 C

FISH

 D

Recombination

Q. 1

Which of these is not a method for introducing genes into a cell?

 A

Transfection

 B

Electroporation

 C

FISH

 D

Recombination

Ans. C

Explanation:

Methods for introducing genes into a cell include, transfection (chemical based), electroporation (physical), infection (viral mediated) and recombination techniques.

 
Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescence-tagged probes to detect specific DNA sequences in tissue samples. It is not used for introducing genes into the cells.
 
Methods for genetic manipulation:
 
Transfection is the most commonly used method for introducing transgenes into human embryonic stem (hES) cells. This system is based on the use of carrier molecules that bind to foreign nucleic acids and introduce them into the cells through the plasma membrane.
 
Electroporation is a method that employs the administration of short electrical impulses that create transient pores in the cell membrane, allowing foreign DNA to enter into the cells. 
 
Infection is gene manipulation by viral infection which can produce a very high percentage of modified cells. Advantage over other systems are, high efficiency of DNA transfer (almost 100% efficiency) and single copy integrations.
Recombination occurs by physical exchange of DNA. An endogenous gene can be replaced by a transfected gene using homologous recombination. Recombination is the result of crossing-over that occurs at chiasmata and involves two of the four chromatids.
 
Ref: Human Embryonic Stem Cell Protocols By Kursad Turksen, 2006, Page 225

Q. 2 Rapid method of chromosome identification in intersex is:

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Q. 2

Rapid method of chromosome identification in intersex is:

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Ans. A

Explanation:

A i.e. FISH
FISH, chromosome painting and spectral karyotyping (SKY) are rapid methods of chromosome identificationQ.


Q. 3

Rapid method of chromosome identification in interphase is –

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Q. 3

Rapid method of chromosome identification in interphase is –

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Ans. A

Explanation:

 
FISH is a cytogenetic technique that can be used to detect the presence or absence of specific DNA sequences (specific gene locus) on chromosomes.
It uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity.
Fluorescence microscopy can then be used to find out where the fluorescent probe bound to the chromosome.
FISH can be used for rapid identification of chromosome during interphase.
FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine cytogenetics or identify extra material of unknown origin.
It can also help in cases where it is difficult to determine from routine cytogenetics if a chromo­some has a simple deletion or is involved in a subtle or complex rearrangement.
In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.

Quiz In Between



Recombinant Dna Technology

RECOMBINANT DNA TECHNOLOGY


RECOMBINANT  DNA  TECHNOLOGY

  • when a gene of one species is transferred to another living organism, it is called recombinant DNA technology(genetic engineering).
  • It involves  isolation  and manipulation  of DNA to  make  chimeric or  hybrid DNA.
  • Chimeric (hybrid) DNA is a recombinant DNA containing genes from two different species, e g. molecule containing both human and bacterial DNA sequences.
  • theres are two distinct techniques for introducing foreign genetic material into plant cell genome.

first through vector which requires:

  1. selection and isolataion of the desirable fragment(s) of DNA which contain gene sequences that need to be cloned (known as insert).
  2. generation of recombinanat DNA (rDNA)  by inserting of dNA fragments into carrier DNA molecules termed as VECTOR.
  3. vector–> bacteria like( Agrobacterium tumefaciens, a virus , a plasmid or any other vector) that replicate within host cell
  4. Introduction of rDNA molecules into host cell, direct introduction involves co-cultivation, microinjection.

Recombinant DNA= Vector+ insert(rDNA)

steps are involved in the construction of recombinant DNA –

  1. Fragmentation of DNA by restriction endonuclease enzyme.
  2. Isolation of specific human DNA sequence.
  3. Insertion of isolated human DNA sequence into vector to form chimeric or hybrid DNA molecule.
  4. joining of two different cut DNA fragments by DNA ligase.

Fragmention  of DNA by restriction  endonuclease:-

  1. Most important  tools  in  genetic  engineering  are  enzymes, nzymes is restriction endonuclease.
  2. These are called so because their presence in a given bacterium restricted the growth of foreign (non-bacterial) DNA, e g. bacteriophages.
  3. These cut  DNA  at specific  DNA  sequence  called  its  restriction  site.
  4. These enzymes recognize certain palindromic sequences in the DNA, ie. the short sequence (4-7 base pairs) which has got two fold rotational symmetry.
  5. a short sequence (4-7 bp) of duplex DNA that is same when two strands are read in opposite direction is called palindrome.
  6. Palindrome serves as the target for most restriction endonucleases, Each restriction enzyme (restriction endonuclease) recognizes specific nucleotide sequence, ie. each restriction enzyme has specific palindromic sequence to recognize
  7. methylation of restriction site prevents host DNA digestionby its own restriction nuclease.
  8. If host cell acquires foreign restriction endonuclease (e.g. byhorizontal transfer i.e. conjugation), host DNA is digested by foreign restriction endonuclease because its restriction site is different, which is not methylated in host DNA.
  9. Some restriction enzymes cut both strand at same level, so as to leave no unpaired bases on either end. These ends are called blunt ends.
  10. restriction enzyme cut two strands in staggered manner, leaving two to four nucleotides of one strand unpaired at each end as (cohesive  ends  or  sticky ends or staggered  ends).

lsolation  of Specific  Human DNA:

  • particular fragment of interest can be separated and isolated by gel electrophoresis (most commonly)

lnsertion  of isolated  human  DNA into  vector

  1. Largest  DNA can  be  incorporated  in  cosmid and  shortest  in  plasmid
  2. larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) and Pt vector.
  3. Plasmid–> pBR 322 < bacteriophase;Lambda charon 4A ( 10-20 kbp) < cosmids (35-50 frp) < BAC = P (50-250 kbp) < YAC (500-3000).
Joining of two  different  cut DNA  fragments:
 
  1. fragments of DNA (one from human genone and another from vector DNA) have base paired, the ends are covalently joined by DNA ligase.

DNA amplification

amplifications of the DNA is required to prepare multiple copies of the DNA, It can be done by: 

  1. Cloning
  2. Polymerase chain reaction (PcR;rrct-our, including real time PCR
  3. Ligase chain reaction (LCR), including Gap LCR
  4. Nucleic acid sequence based amplification (NASBA)

Method  (ii),  (iii)  and  (iv)  are  also  called, nucleic  acid amplification  tests  (NAAT)

Exam Important

  1. It involves  isolation  and manipulation  of DNA to  make  chimeric or  hybrid DNA.\
  2. generation of recombinanat DNA (rDNA)  by inserting of dNA fragments into carrier DNA molecules termed as VECTOR.
  3. vector–> bacteria like( Agrobacterium tumefaciens, a virus , a plasmid or any other vector) that replicate within host cell.
  4. joining of two different cut DNA fragments by DNA ligase.
  5. Palindrome serves as the target for most restriction endonucleases, Each restriction enzyme (restriction endonuclease) recognizes specific nucleotide sequence, ie. each restriction enzyme has specific palindromic sequence to recognize.

amplifications of the DNA is required to prepare multiple copies of the DNA, It can be done by: 

  1. Cloning
  2. Polymerase chain reaction (PcR;rrct-our, including real time PCR
  3. Ligase chain reaction (LCR), including Gap LCR
  4. Nucleic acid sequence based amplification (NASBA)
Don’t Forget to Solve all the previous Year Question asked on RECOMBINANT DNA TECHNOLOGY

Module Below Start Quiz

Recombinant Dna Technology

RECOMBINANT DNA TECHNOLOGY

Q. 1 Which of the following statements describing restriction endonucleases is TRUE?

 A

They always yield blunt ends

 B

They recognize methylated DNA sequence

 C

They recognize triplet repeats

 D

They cleave both strands in duplex DNA

Q. 1

Which of the following statements describing restriction endonucleases is TRUE?

 A

They always yield blunt ends

 B

They recognize methylated DNA sequence

 C

They recognize triplet repeats

 D

They cleave both strands in duplex DNA

Ans. D

Explanation:

Restriction endonucleases are produced by prokaryotes for cleaving both strands of foreign DNA.
The host cell’s DNA is not degraded because the recognition sites are specifically methylated.
The endonucleases recognize specific short symmetrical sequences known as palindromes.
These cleavage sites contain two fold rotational symmetry in that the sequence is identical but antiparallel in the complementary strands.

In some cases, single-stranded cohesive ends on each of the complementary strands are produced, while in other cases double-stranded blunt ends are formed.
Modern analysis of DNA structure is highly dependent upon the use of different restriction endonucleases that permit the specific hydrolysis of DNA into large polynucleotides. 

Ref: Weil P. (2011). Chapter 39. Molecular Genetics, Recombinant DNA, & Genomic Technology. In D.A. Bender, K.M. Botham, P.A. Weil, P.J. Kennelly, R.K. Murray, V.W. Rodwell (Eds), Harper’s Illustrated Biochemistry, 29e.

Q. 2 After digestion by restriction endonucleases DNA strands can be joined again by which of the following enzymes?

 A

DNA polymerase

 B

DNA ligase

 C

DNA topoisomerase

 D

DNA gyrase

Q. 2

After digestion by restriction endonucleases DNA strands can be joined again by which of the following enzymes?

 A

DNA polymerase

 B

DNA ligase

 C

DNA topoisomerase

 D

DNA gyrase

Ans. B

Explanation:

DNA is cleaved into fragments by restriction endonucleases. After the fragments of DNA have base paired, the ends are covalently joined by the action of DNA ligase

Restriction enzymes in conjunction with DNA ligase can produce vector containing recombinant or hybrid or chimeric DNA.
 
Ref: Essentials of Biochemistry By Naik, 2012, Page 335.

Q. 3 DNA fragments formed by the action of Restriction Endonucleases, are separated by:

 A

Gel electrophoresis

 B

Agarose gel eletrophoresis

 C

Paper Chromatography

 D

High pressure liquid chromatography

Q. 3

DNA fragments formed by the action of Restriction Endonucleases, are separated by:

 A

Gel electrophoresis

 B

Agarose gel eletrophoresis

 C

Paper Chromatography

 D

High pressure liquid chromatography

Ans. B

Explanation:

B i.e. Agarose gel electrophoresis

Restriction endonuclease (restriction enzyme) makes 2 incisions through sugar phosphate backbone (phospho diester bond) of each strand of ds DNAQ at specific sequence k/a recognition sequences or restriction site. So RE restricts viral (bacteriophage – DNA) replicationQ and protects bacteria (prokaryote) from infection by virusQ.

RE cuts both strands of double stranded (ds) DNA at specific restriction sites with palindromic (inverse repeat) arrangement; thus producing smaller, manageable fragments with short sequenes and sticky/ blunt endsQ. These restriction fragments can be isolated by agarose gel /polyacrylamide – electrophoresisQ or HPLC.

After digestion by restriction endonucleases the DNA ends can be ligated (joined/ annealed) by ‘ DNA-ligaseQ

Quiz In Between


Q. 4

In DNA transfer the vectors used from smallest to largest is:

 A

Cosmids, Plasmids, Bacteriophage

 B

Plasmids, Bacteriophage, Cosmids

 C

Bacteriophage, Cosmides, Plasmids

 D

Cosmids, Bacteriophage, Plasmids

Q. 4

In DNA transfer the vectors used from smallest to largest is:

 A

Cosmids, Plasmids, Bacteriophage

 B

Plasmids, Bacteriophage, Cosmids

 C

Bacteriophage, Cosmides, Plasmids

 D

Cosmids, Bacteriophage, Plasmids

Ans. B

Explanation:

B i.e. Plasmid, bacteriophage, cosmid 

*       Plasmid is smallest & most commonly used vectors

*       1Kb = 1000 nucleotide long base

Vector/ Vehicle- DNA

DNA insert size (i.e. can accept DNA fragment of)

Plasmid (PBR 322)

0.01 – 10 kb (smallest)(2

Bacteriophage

(Lambda charon 4A)

10 -20 kb

Cosmids

35 – 50 kb (largest)2


Q. 5 Palindrome is associated with:        

 A

Synthesis of DNA

 B

Extrachromosomal molecule of DNA

 C

Sequence of DNA

 D

Small nuclear RNA

Q. 5

Palindrome is associated with:        

 A

Synthesis of DNA

 B

Extrachromosomal molecule of DNA

 C

Sequence of DNA

 D

Small nuclear RNA

Ans. C

Explanation:

 

Palindromic DNA – A palindrome is a sentence that reads the same forwards and backwards, e.g. ‘Madam I’m Adam’. The DNAs of several eukaryotes are shown to have palindromic sequences, in which nucleotides of one strand going in one direction are the same as the nucleotides of the other strand going in the other direction.

The exact significance of palindromic DNA is not known, although several functions have been suggested.

Short palindromes may function as recognition sites of DNA for proteins which also have a two-fold rotational symmetry, e.g. lac repressor protein, CRP protein and many bacterial restriction enzymes.

Palindromes may also give structural strength to the transcribed RNA by hydrogen bonding in the hairpin loops. If the palindromic sequences are not perfectly symmetrical, imperfect loops may result.

Quiz In Between


Q. 6

DNA amplification is done in by all, except:   
PGI 06

 A

Polymerase chain reaction

 B

Nucleic Acid Sequence Based Amplification

 C

Ligase chain reaction

 D

DNA sequencing

Q. 6

DNA amplification is done in by all, except:   
PGI 06

 A

Polymerase chain reaction

 B

Nucleic Acid Sequence Based Amplification

 C

Ligase chain reaction

 D

DNA sequencing

Ans. D

Explanation:

Ans. DNA sequencing


Q. 7

Nucleic acid amplification techniques are:

 A

PCR

 B

Real time PCR

 C

DNA Cloning

 D

Next generation DNA sequencing

Q. 7

Nucleic acid amplification techniques are:

 A

PCR

 B

Real time PCR

 C

DNA Cloning

 D

Next generation DNA sequencing

Ans. A:B

Explanation:

Ans: a. PCR …, b. Real time ….[Ref Harper 30th/458; Robbins 9th/180; Lippincott 6th/479;Harrison 19th/ 150e-7; http://link. springer. corn]

  • Real-time PCR automates the laborious process of amplifica­tion by quantitating reaction products for each sample in every
  • Cycle.
  • There are several methods for amplification (copying) of small numbers of molecules of nucleic acid to readily detectable levels.
  • These NAATs include PCR, LCR, strand displacement amplification, and self-sustaining sequence replication.
  • The amplified nucleic acid can be detected after the reaction is complete or (in real-time detection) as amplification proceeds. The sensitivity of NAATs is far greater than that of traditional assay methods such as culture.

Quiz In Between



REAL TIME PCR

REAL TIME PCR


Real Time PCR- Principle, Process, Markers, Advantages, Applications

  • Real Time PCR is a technique used to monitor the progress of a PCR reaction in real time.
  • At the same time, a relatively amount of PCR product (DNA, cDNA or RNA) can be quantified.
  • Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds.
  • A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).
  • Conventional PCR is a powerful technique that allows exponential amplification of DNA sequences.
  • A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase.
  • The copies produced after the extension, so called amplicons, are re-amplified with the same primers leading thus to an exponential amplification of the DNA molecules.
  • After amplification, however, gel electrophoresis is used to analyse the amplified PCR products and this makes conventional PCR time consuming; since the reaction must finish before proceeding with the post-PCR analysis. Real Time PCR overcomes this problem.
  • The term “real time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.

Principle of Real Time PCR

This same principle of amplification of PCR is employed in real-time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real-time”. The reaction is placed in to a real-time PCR machine that watches the reaction occur with a camera or detector.

RT-PCR can be done in two ways.

  1. One step RT-PCR
  2. Two step RT-PCR

One step

RT-PCR, both reverse transcription and DNA amplification happen in a single tube. First, at 44°C the ssRNA, specific primers and Reverse transcriptase are used to form dsDNA and then as the temperature is increased to 94°C, the reverse transcriptase is inhibited and the primers and DNA polymerase act to form multiple copies of dsDNA

two step

  • RT-PCR, in the first tube, Reverse transcriptase and non specific primers like oligodTs or random hexamers are used. This step converts any RNA that is present in the sample to dsDNA. This step is carried out at 44°C. This is transferred to another tube into which specific primers, all the four dNTPs and DNA polymerase are added. This is subjected to thermocycling (denaturation at 94°C, Annealing at Tm- 5 and elongation at 72°C) to get multiple copies of DNA.

Advantages of Real Time PCR

  • It gives a look in to the reaction that is help to decide which reactions have worked well and which have failed.
  • The efficiency of the reaction can be precisely calculated.
  • There is no need to run the PCR product out on a gel after the reaction as the melt curve analysis serve the purpose.
  • The real-time PCR data can be used to perform truly quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semi-quantitative at best.
  • Faster than normal PCR.
  • Less complexity at the quantification of sample.etc

Applications of Real Time PCR

  • Gene expression analysis
    • Cancer research
    • Drug research
  • Disease diagnosis and management
    • Viral quantification
  • Food testing
    • Percent GMO food
  • Animal and plant breeding
    • Gene copy number

As expected the Advantage of Two step RT-PCR is that because the first step uses non specific primers, we can amplify many genes by using more sets of specific primers in the second step. Disadvantage is that it involves multiple steps and hence the workflow is complex and the source of contamination of the sample is higher.

RT-PCR on the whole has few disadvantages -It is not sensitive and not specific. The amplification is not adequate. Requires expensive thermocycling equipment, To overcome these difficulties we use NASBA

NASBA

  • It is a reaction consists of avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase and RNase H with two oligonucleotide primers.
  • The steps involved are:
  • The RNA strand present in the sample acts as a sense RNA. To this sense RNA, primer P1 binds (synthesised in such a way that it is complementary to one end of RNA)
  • AMV reverse transcriptase elongates the DNA strand complementary to sense RNA to form RNA:DNA hybrid
  • RNAase H removes RNA part of RNA:DNA hybrid
  • P2 primer binds to the other end of the new DNA strand
  • AMV reverse transcriptase elongates the DNA strand from P2 primer forming a ds DNA
  • P1 primer is so synthesised that once it forms a dsDNA, it acts as a promotor for T7 RNApolymerase. So, once dsDNA is formed, T7 RNA polymerase binds to P1 primer site and it starts transcribing the dsDNA to form multiple copies of RNAs.
  • These antisense RNAs now act as templates for primer P2 and reverse transcriptase acts on it
  • That forms dsDNA and the entire cycle is repeated so that the amplification is more than 1012 fold in 90 to 120 minutes.
  • The whole process happens at 41 °C as there is no need for a denaturation step. Reason being, NASBA uses RNA polymerase and not DNA polymerase

Advantages of NASBA over RT-PCR:

  • The amplification of nucleic acid sequence of more than 109 copies can be done in just 90 minutes by the three-enzyme action.
  • Expensive thermocycling equipment is not needed as the reaction occurs isothermally at 41°C.
  • It helps in better RT-PCR reaction as it offers faster amplification kinetics.

Exam Important

  • Conventional PCR is a powerful technique that allows exponential amplification of DNA sequences.
  • A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase.
  • The term “real time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.
  • it is used in Disease diagnosis and management, and Viral quantification
  • Zika virus is the example for RT PCR, most sensitive for HIV.
Don’t Forget to Solve all the previous Year Question asked on REAL TIME PCR

Module Below Start Quiz

REAL TIME PCR

Real Time PCR

Q. 1 Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Q. 1

Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Ans. D

Explanation:

Ans. (d) To know how much amplification of DNA has occured PF 1,m,etz. 25/e 714, 27/e, p 124; Greenwood 18/e 79

Real Time. PCR

  • It is the molecular detection technique that discriminates real time amplification from conventional PCR assays.
    • The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction.
    • This combines the DNA amplification and detection steps into one homogeneous assay and obviates the requirement for gel electrophoresis to detect amplification products.
    • Its simplicity, specificity, and sensitivity, together with its, more reliable instrumentation, and improved protocols, has made realtime                     oenLhmark technology for lite ocLuLtion of DNA.
    • Real time PCR is extremely useful in medical microbiology, with greatest impact on virology.

Q. 2 Which of the following is most sensitive for diagnosis of HIV?

 A

RT PCR

 B

bDNA assay

 C

NASBA

 D

P24 detection

Q. 2

Which of the following is most sensitive for diagnosis of HIV?

 A

RT PCR

 B

bDNA assay

 C

NASBA

 D

P24 detection

Ans. A

Explanation:

Ans. a. RT PCR


Q. 3

Which of the following techniques is based on RNA?

 A

RT PCR

 B

Sanger’s technique

 C

Next generation sequencing

 D

Western blot

Q. 3

Which of the following techniques is based on RNA?

 A

RT PCR

 B

Sanger’s technique

 C

Next generation sequencing

 D

Western blot

Ans. A

Explanation:

Ans: A. RT PCR
Ref: Harper’s illustrated biochemistry,3Oh ed, pg. 29,457 and Tietz Fundamental of clinical chemistry and molecular diagnostics, Vh ed.
RT-PCR:

  • Reverse transcription polymerase chain reaction (RTPCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR).
  • It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
  • A method used to quantitate mRNA levels that rely upon a first step of cDNA copying of mRNAs catalysed by reverse transcriptase prior to PCR amplifi cation and quantitation.

 Sanger sequencing,

  • Also known as the chain termination method, is a technique for DNAsequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.

Quiz In Between


Q. 4

In which of the following, viral load done by Real Time PCR is of no role in investigative procedures?

 A

Person with hepatitis B on tenofovir therapy

 B HSV causing temporal encephalitis

 C

BK virus in patient of allograft renal transplant

 D

CMV PCR in blood of patient of liver transplant

Q. 4

In which of the following, viral load done by Real Time PCR is of no role in investigative procedures?

 A

Person with hepatitis B on tenofovir therapy

 B

HSV causing temporal encephalitis

 C

BK virus in patient of allograft renal transplant

 D

CMV PCR in blood of patient of liver transplant

Ans. B

Explanation:

Ans. B. HSV causing temporal encephalitis
Ref: “Diagnosis ofherpesvirus infections ofthe central nervous system.’, Herpes : the journal of the IHMF I1 Suppl 2 2004, pp. 48A-564.

  • The question simply asks in which of the given conditions calculation of Viral load is not required. In HSV causing temporal encephalitis the role of PCR is just to detect HSV DNA and make a diagnosis of the disease.
  • There is no role of detection of the viral load copies in the management or diagnosis of the disease.



Q. 5

Best assessment of protein binding regions on a DNA molecule can be done by:

 A

DNA footprinting

 B

RT PCR

 C

Microarray

 D

Western blotting

Q. 5

Best assessment of protein binding regions on a DNA molecule can be done by:

 A

DNA footprinting

 B

RT PCR

 C

Microarray

 D

Western blotting

Ans. A

Explanation:

Answer-(a) DNA footprinting [Ref: www.biotecharticles.com; www.biologyexams4u.com Lippincott 6th/473]

  • DNA footprinting- An in-vitro technique to find out protein binding regions on a DNA molecule. The technique is also called as DNAse I footprinting.Thousands of proteins (enzymes) are interacting with DNA in the nucleus for regulating activities like replication, transcription, translation etc.
  • DNA Footprinting is a molecular technique used to identify the specific DNA sequence (binding site) that binds to a protein.
  • This technique mainly used to identify the transcription factors which bind to promoter, enhancer or silencer region of gene to regulate its expression. Therefore the regulation of transcript ion of a gene can be studied using this method.

Q. 6

True about ZIKA virus:

 A Belong to flavivirus

 B

First case detected in 1953 in Nigeria

 C

RT PCR is useful in diagnosis

 D

Causes macrocephaly

Q. 6

True about ZIKA virus:

 A

Belong to flavivirus

 B

First case detected in 1953 in Nigeria

 C

RT PCR is useful in diagnosis

 D

Causes macrocephaly

Ans. A:C:E

Explanation:

Answer: (a) Belong to flavivirus, (c) RT PCR is useful in diagnosis, (e) May presents with conjunctivitis (Ref: Harrison 19th/1314; www. cdc. gov;www. nytimes. corn] 

  • It is spread mostly by the bite of an infected Aedes species mosquitoes (A. aegptiand A. albopictus). T
  • It can be passed to a pregnant woman to her fetus. Infection during pregnancy can cause certain birth defects..
  • Real-time reverse transcription-polymerase chain reaction (RT PCR) testing should be performed on serum collected during the first two weeks after symptom onset.
  • There’s no vaccine or specific treatment tor tlw disease. Treatment instead focuses on relieving symptoms and includes rest, rehydration and medications for fever and pain.
  • A maculopapular rash, conjunctivitis, myalgia, and arthralgia usually accompany or follow those manifestations.

Quiz In Between



Difference between Meningitis and Encephalitis

Difference between Meningitis and Encephalitis


S.N.

Character

Meningitis

Encephalitis

1. Definition Inflammation of the thin membranes that surround the brain and spinal cord called the meninges (mostly of the pia mater and arachnoid mater). Inflammation of the brain parenchyma.
2. Causative Agent Bacteria, Viruses and Fungi Mostly Viruses
3. Microorganisms involved The most common are echovirus, poliovirus, and coxsackie.

Bacterial meningitis develops under the action of StreptococcusNeisseriaHaemophilusListeria, and other bacteria.

Viruses (herpes virus, rabies virus, arboreal viruses, cytomegalovirus, etc.);

Bacteria – often occurring as meningoencephalitis caused by meningococci, pneumococci,

Hemophilus, tuberculous bacteria, etc.)

Fungi or parasites such as leptospirosis, toxoplasmosis, trichinellosis, etc.

4. Other Causes
  • Response to a brain tumor;
  • Reaction after a chemotherapy;
  • Poisoning with lead;
  • Reaction after complex studies with a contrast medium;
  • Parasites, fungal infections.
  • Complicated brain tumors;
  • Sarcoidosis;
  • Leukemia;
  • Multiple sclerosis;
  • Lead poisoning;
  • Reaction after application of various substances in the liquor;
  • Cerebrovascular accidents, etc.
5. Incubation period The symptoms of meningitis develop within a few hours to a couple of days. The most common incubation period is 3-5 days.
6. Symptoms Headache is most common, along with vomiting or nausea, skin rash or discoloration of skin, high fever, stiff neck, confusion, double vision. Headache, joint pain, irritability, fever, lethargy, seizures, behavioral changes.
7. Complications
  • Temporary or permanent loss of hearing or vision;
  • Irreversible brain damage, affecting the cognitive abilities, movement abilities
  • Hydrocephalus;
  • Encephalitis.
  • Memory problems
  • Personality and behavioral changes
  • Speech problems
  • Epilepsy
  • Physical and motor difficulties
  • Low mood
  • Fatigue
8. Rash Skin discoloration or rash  may be present Absent
9. Altered Mental Status No focal deficits or AMS Altered Mental Status
10. Forms Meningitis can be only of one form. Primary and secondary encephalitis.

  • Primary: the brain and spinal cord are directly affected.
  • Secondary: the infection enters the brain after affecting another organ.
11. Types
  • Tuberculous meningitis,
  • Aseptic meningitis
  • Syphilitic aseptic meningitis
  • Cryptococcal meningitis
  • Staphylococcal meningitis
  • Gram negative meningitis
  • Pneumococcal meningitis
  • H.influenza meningitis Meningococcal meningitis.
  • Polyoencephalitis – localized in the gray matter of the brain/spinal cord;
  • Leucoencephalitis – localized in the cerebral white brain matter;
  •  Panencephalomyelitis – localized in the gray and white matter of the brain/spinal cord;
  • Perivenous encephalomyelitis.
12. Modes of Transmission

(Transmission possibility depends on the type of causative agent involved)

Bacteria are present in discharges from the nose and mouth and cause transmission through droplets.
  • Breathing in respiratory droplets from an infected person
  • Skin contact
  • Mosquito, tick, and other insect bites
  • Tick of Horses
  • Migratory Birds
  • Contaminated food or drink
13. Risk group age Those older than 60 and younger than 5 are at highest risk. Occurs most frequently in infants younger than 1 year of age and in elderly patients older than the age of 65 with intermediate incidence in individuals between these age extremes.
14. Diagnosis Physical examination,

Followed by CBC with di?erential, C-reactive protein, and blood for Gram stain and culture.

Clinical presentation and supported by spinal ?uid analysis and neuroimaging abnormalities, CSF, PCR.
15. CSF findings The cerebrospinal fluid (CSF) is abnormal. The CSF findings are variable.
16. Treatment Ampicillin is usually prescribed along with an aminoglycoside or a cephalosporin (cefotaxime) medication. Acyclovir, and is usually administered intravenously in the hospital for at least ten days.
17. Vaccines Vaccines of varying effectiveness exist against the following bacterial causes of meningitis:

  • Streptococcus pneumoniae;
  • Neisseria meningitides;
  • Mycobacterium tuberculosis.
Vaccines exist against some types of encephalitis:

  • Tick-borne encephalitis vaccine;
  • Japanese encephalitis vaccine.

Exam Important

  • Bacterial meningitis develops under the action of StreptococcusNeisseriaHaemophilusListeria, and other bacteria.
  • Bacteria are present in discharges from the nose and mouth and cause transmission through droplets.
  • Mosquito, tick, and other insect bites,  Tick of Horses,  Migratory Birds,  Contaminated food or drink causes Encephalitis
  • A mpicillin is usually prescribed along with an aminoglycoside or a cephalosporin (cefotaxime) medication. in  meningitis 
  • Acyclovir, and is usually administered intravenously in the hospital for at least ten days in Encephalitis.

 

Don’t Forget to Solve all the previous Year Question asked on Difference between Meningitis and Encephalitis

Module Below Start Quiz

Difference between Meningitis and Encephalitis

Difference between Meningitis and Encephalitis

Q. 1 Drug used to treat Cryptococcal meningitis is:
March 2007

 A

Pentostatin

 B

Amphotericin B

 C

Zidovudine

 D

Clotrimazole

Q. 1

Drug used to treat Cryptococcal meningitis is:
March 2007

 A

Pentostatin

 B

Amphotericin B

 C

Zidovudine

 D

Clotrimazole

Ans. B

Explanation:

Ans. B: Amphotericin B

Cryptococcus neoformans is an encapsulated yeast-like fungus. It causes meningitis, especially as a secondary infection in AIDS patients.

Cryptococcosis that does not affect the central nervous system can be treated with fluconazole alone.

Cryptococcal meningitis should be treated for two weeks with intravenous Amphotericin B and oral flucytosine. Main disadvantage of Amphotericin B includes nephrotoxicity.


Q. 2 Most common cause for meningitis in adults:
September 2009

 A

H.Influenzae

 B

N.meningitidis

 C

Staph.aureus

 D

Streptococcus pneumoniae

Q. 2

Most common cause for meningitis in adults:
September 2009

 A

H.Influenzae

 B

N.meningitidis

 C

Staph.aureus

 D

Streptococcus pneumoniae

Ans. D

Explanation:

Ans. D: Streptococcus pneumoniae

Streptococcus pneumoniae is the most common cause of meningitis in adults of age over twenty years, accounting for nearly half of the cases.

N. meningitidis accounts for nearly 25% of the cases. Staph.aureus and coagulase negative staphylococci are important causes of meningitis that occurs following neurosurgical procedures.


Q. 3 Most common causative agent for meningitis in the immunocompromised patient is:      
September 2009

 A

Histoplasmosis

 B

Cryptococcus

 C

Candida albicans

 D

Coccidiomycosis

Q. 3

Most common causative agent for meningitis in the immunocompromised patient is:      
September 2009

 A

Histoplasmosis

 B

Cryptococcus

 C

Candida albicans

 D

Coccidiomycosis

Ans. B

Explanation:

Ans. B: Cryptococcus

Cryptococcosis is a systemic or central nervous system (CNS) fungal infection caused by the yeast Cryptococcus neoformans. The organism is ubiquitous, but is particularly plentiful in soils enriched with bird droppings.

In immunocompetent patients, cryptococcal infection is usually asymptomatic, self-limited, and confined to the lungs.

In persons with advanced HIV infection (e.g., those with CD4 counts

In HIV-infected patients, Cryptococcus can infect almost all organs in the body, but most commonly causes meningitis or meningoencephalitis.

Quiz In Between


Q. 4 Drug treatment is given for how many days in pneumococcal meningitis

 A

5 days

 B

7 days

 C

14 days

 D

21 days

Q. 4

Drug treatment is given for how many days in pneumococcal meningitis

 A

5 days

 B

7 days

 C

14 days

 D

21 days

Ans. C

Explanation:

Ans. is ‘c’ i.e., 14 days

Recommendations for duration of treatment

  • Pneumococcal meningitis                       —> 10-14 days
  • Meningococcal meningitis                              5-7 days
  • Hib meningitis                                       —> 7-14 days
  • Listeria meningitis                                 —>     21 days

Q. 5

How long should a child be isolated after being diagnosed with bacterial meningitis to prevent further transmission?

 A Till 24 hours after starting antibiotics

 B

Till cultures become negative

 C

Till antibiotic course is complete

 D

Till l2hrs alter admission

Q. 5

How long should a child be isolated after being diagnosed with bacterial meningitis to prevent further transmission?

 A

Till 24 hours after starting antibiotics

 B

Till cultures become negative

 C

Till antibiotic course is complete

 D

Till l2hrs alter admission

Ans. A

Explanation:

Ans: A. Till 24 hours after starting antibiotics
Ref: Ghai Essentisl Pediatrics, 8’t’ ed,, pg. 565′ https://www.cdc.gov
Prevention of transmission:

  • Droplet precautions for the first 24 hours of antimicrobial therapy is sufficient

Quiz In Between



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