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Lecture 3 PHBC731

Mohamed Zakaria Gad

Prof. of Biochemistry

Mohamed.gad@guc.edu.eg

Molecular Biology TechniquesPart 1

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Learning Learning ObjectivesObjectivesThis lecture reviews some of the techniques used in This lecture reviews some of the techniques used in molecular biology; the methods that concern molecular biology; the methods that concern themselves with analysis of the structure & function themselves with analysis of the structure & function of DNA & RNA at the molecular level. of DNA & RNA at the molecular level.

These techniques are important in:These techniques are important in:

• Diagnosis of genetic disorders Diagnosis of genetic disorders (e.g. cystic fibrosis).(e.g. cystic fibrosis).

• Diagnosis of certain infective disorders Diagnosis of certain infective disorders (e.g. human (e.g. human

immunodeficiency virus (HIV) and hepatitis B & C infections). immunodeficiency virus (HIV) and hepatitis B & C infections).

• Investigation of the molecular basis of cancer. Investigation of the molecular basis of cancer. • Studying common polygenic disorders (e.g. Studying common polygenic disorders (e.g.

essential hypertension and diabetes mellitus).essential hypertension and diabetes mellitus).

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Techniques of Techniques of Molecular BiologyMolecular Biology

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Polymerase Chain Reaction (PCR)

Application: in vitro amplification of DNA segments, without using a living organism (such as E. coli or yeast).

Invented in 1993 by Kary Mullis Received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR).

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PCR Revolutionized the Study of PCR Revolutionized the Study of GenesGenes• PCR allows a small section of human genomic DNA

to be amplified many times in the test tube.

• The technique is simple, fast, and requires as little as a single cell.

• DNA for analysis may be obtained from any nucleated cell; most commonly used are WBCs, but hair roots, mouth scrapings, or sperm can also be used.

• Great care must be taken to avoid contamination with cells or DNA from the laboratory.

• Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of up to 40 kb.

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PCR in PCR in PracticePracticeBasic components are:

1. DNA template that contains the region of the DNA fragment to be amplified

2. Primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.

3. DNA polymerase (e.g. Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C), used to synthesize a DNA copy of the region to be amplified

4. Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA

5. Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase

6. Divalent cation, magnesium or manganese ions; generally Mg2+ is used

7. Monovalent cation potassium ions

The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. The PCR usually consists of a series of 20 to 35 cycles.

PCR ( Polymerase chain reaction)How to magnify a desired gene?

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Ethidium bromide-stained PCR Ethidium bromide-stained PCR products after Gel electrophoresis. products after Gel electrophoresis. 2 sets of primers were used to 2 sets of primers were used to amplify the IGF gene from 3 amplify the IGF gene from 3 different DNA samples. In sample different DNA samples. In sample #1 the gene was not amplified by #1 the gene was not amplified by PCR, bands for tissue #2 and #3 PCR, bands for tissue #2 and #3 indicate successful amplification of indicate successful amplification of the IGF gene. A +ve control, and a the IGF gene. A +ve control, and a DNA ladder containing DNA DNA ladder containing DNA fragments of defined length (last fragments of defined length (last lane to the right) to estimate lane to the right) to estimate fragment sizes in the experimental fragment sizes in the experimental PCRs, were also ran on this gel.PCRs, were also ran on this gel.

How to Express How to Express Results ?Results ?To check whether the PCR generated the anticipated DNA fragment, agarose gel electrophoresis is commonly employed for size separation of the PCR products. The size(s) of PCR products is thereby determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel alongside the PCR products

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Practical modifications to PCR Practical modifications to PCR techniquetechnique

RReverse everse TTranscription PCR (RT-PCR)ranscription PCR (RT-PCR)

A method used to amplify, isolate or identify a known sequence from a cellular or tissue RNARNA. PCR reaction is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript. RT-PCR reaction is widely used in the diagnosis of genetic diseases.

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenza virus A and retroviruses like HIV.

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Real Time PCR Real Time PCR

This technique enables both detection and quantification of one or more specific sequences in a DNADNA sample. It is a kinetic approach in which you look at the reaction in the early stages while it is still linear .

The principle is based on a DNA-binding dye (e.g. SYBR Green) binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified

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Clinical Applications of Clinical Applications of PCRPCR1.1. Allow the amplification & analysis of the DNA in Allow the amplification & analysis of the DNA in

a single cell, hair follicle, sperm, … to serve a single cell, hair follicle, sperm, … to serve investigations in investigations in forensic medicineforensic medicine..

2.2. Detection of Detection of infectious agentsinfectious agents, especially latent , especially latent viruses. viruses.

3.3. Prenatal genetic diagnosisPrenatal genetic diagnosis of mutations. of mutations.

4.4. Detection of Detection of allelic polymorphismallelic polymorphism..

5.5. Establishing Establishing precise tissue typesprecise tissue types for transplants. for transplants.

6.6. Study of Study of evolutionevolution, using DNA from , using DNA from archaeological sample.archaeological sample.

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Southern Blotting

Application: Allows detection of DNA fragments by hybridisation to a suitable probe.

Named after Dr. Edward M. Southern who developed this procedure at Edinburgh University in the 1970s

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Procedure:

1) DNA is cut first with restriction endonucleases.

2) The cleaved DNA fragments are separated on the basis of size using agarose gel electrophoresis.

3) After alkaline denaturation, the DNA fragments are transferred to a nitrocellulose membrane (the blot), by layering the nitrocellulose membrane over the gel (which rests in buffer). The gel is placed on adsorbent paper which acts as a wick to draw buffer through the gel and enhance DNA transfer to the membrane. The membrane is much easier to handle than the fragile agarose.

4) The DNA can be analysed with labelled probes that will hybridise to complementary sequences.

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Clinical Applications of Southern Clinical Applications of Southern BlottingBlotting1.1. Detection of presence & determination of the Detection of presence & determination of the number of copies number of copies

of particular sequence or geneof particular sequence or gene in genomic DNA, e.g. determine in genomic DNA, e.g. determine if liver has a gene for insulin? or how many copies of LDL if liver has a gene for insulin? or how many copies of LDL receptor gene are present in the liver of heterozygote FH receptor gene are present in the liver of heterozygote FH patient as compared to normal individual?patient as compared to normal individual?

2.2. Diagnosis of Diagnosis of single-gene defectssingle-gene defects. e.g. in sickle-cell anaemia, . e.g. in sickle-cell anaemia, the single a.a substitution of valine for glutamic acid is the the single a.a substitution of valine for glutamic acid is the result of a single-base change in which thymine replaces result of a single-base change in which thymine replaces adenine. This single base change leads to loss of recognition adenine. This single base change leads to loss of recognition sequence for restriction endonuclease termed MstIII in the sequence for restriction endonuclease termed MstIII in the mutant gene. When the genomic DNA is cut with this enzyme mutant gene. When the genomic DNA is cut with this enzyme and subjected to southern blotting using a probe specific for the and subjected to southern blotting using a probe specific for the -globin gene, the length of DNA containing the complementary -globin gene, the length of DNA containing the complementary sequence differs between the sickle cell and normal sequence differs between the sickle cell and normal specimens.specimens.

Similar to Southern blot, Northern (RNA) and Western (protein) blots are used to size and quantitate specific RNA and protein molecules, respectively.

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SNPs, Pronounced “snips”, are single nucleotide polymorphisms or one-letter variations in the DNA sequence, mostly in non protein-coding regions of the genome. They occur about every 1250 bases. SNPs contribute to differences among individuals. The majority have no effect, others cause subtle differences in countless characteristics, like appearance, while some affect the risk for certain diseases.

Single Nucleotide

Polymorphisms (SNPs)

and Differences in

Humans

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Restriction Fragment Length Polymorphism

(DNA Fingerprinting) (RFLP Analysis)

Application: Identify a change in the genetic sequence that occurs at a site where a restriction enzyme cuts.

Restriction enzymes are proteins isolated from bacteria that recognize specific short sequences of DNA and cut the DNA at those sites. The normal function of these enzymes in bacteria is to protect the organism by attacking foreign DNA, such as viruses.

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Procedure

Restriction enzyme is added to the DNA being analyzed and incubated for several hours, allowing the restriction enzyme to cut at its recognition sites. The DNA is then run through a gel, which separates the DNA fragments according to size. You can then visualize the size of the DNA fragments and assess whether or not the DNA was cut by the enzyme.

Result

The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals, due to insertions, deletions or transversions. This causes the length of the fragments to vary between individuals (thus polymorphism).

eNOS Glu298Asp (G→T at nucleotide 894, exon 7) gene polymorphism

(A real work from our department)

137 bp

320 bp

457 bp

TTGG

A PCR-RFLP using BAN II for the identification of G (Glu) and T (Asp) alleles. BAN II cuts the site if G is present, not if T. Since we have two genes on two chromosomes for eNOS (endothelial nitric oxide synthase), so the possibilities for this site at the two chromosomes will be either GG, GT, or TT. This will appear as 1 band for TT (457 bp, no cut), 2 bands for GG (at 320 bp & 137 bp), and 3 bands for GT (457 bp, 320 bp & 137 bp).

GT

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Clinical Applications of Clinical Applications of RFLPRFLP1.1. Trace inheritance patterns; it can show the Trace inheritance patterns; it can show the

genetic relationship between individuals, genetic relationship between individuals, because children inherit genetic elements because children inherit genetic elements from their parents. from their parents. Mitochondrial DNA Mitochondrial DNA RFLP analyses can lead to the RFLP analyses can lead to the determination of maternal relationships. determination of maternal relationships.

2.2. Identify specific mutations. Identify specific mutations.

3.3. Determine relationships among and Determine relationships among and between species.between species.

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SOUTHERN.EXE

Investigation of A Crime Investigation of A Crime (RAPING) Using PCR , RFLP (RAPING) Using PCR , RFLP

& Southern blotting & Southern blotting TechniquesTechniques

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Based on the technique of hybridization:

1. HCV is a single-stranded RNA virus in the Flaviviridae family. The genome is approximately 10,000 nucleotides and encodes a single polyprotein of about 3,000 amino acids.

2. If an individual is infected with a virus (e.g. HCV), the genetic material of the virus will be different from the individual’s DNA or RNA.

3. RNA (including viral RNA) is isolated from patient’s blood.

4. Detect viral RNA by RT-PCR using primers targeting the HCV sequence.

Detection of Hepatitis C virus Detection of Hepatitis C virus (HCV) infection(HCV) infection

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Glossary: “Molecular Biology Language”Autoradiography: The detection of radioactive molecules (e.g DNA, RNA, protein) by visualization of their effects on photographic film.Bacteriophage: a virus that infects a bacterium.Blunt-ended DNA: Two strands of a DNA having ends that ae flush with each other.cDNA: A single-sranded DNA molecule that is complementary to a mRNA molecule and is synthesized from it by the action of reverse transcriptase.Chimeric molecule: A molecule (e.g. DNA, RNA, protein) containing sequences derived from two different species.Clone: A large number of cells or molecules that are identical with a single parental cell or molecule.Endonuclease: An enzyme that cleaves internal bonds in DNA or RNA.Exon: The sequence of a gene that is expressed immature mRNA.Exonuclease: An enzyme that cleaves nucleotides from either the 3’ or 5’ ends of DNA or RNA.Hybridization: The specific association of complementary strands of nucleic acids.Insert: An additional length of base pairs in DNA, generally introduced by the techniques of recombinant DNA technology.Intron: The sequence of a gene that is transcribed but excised before translation.Library: A collection of cloned fragments that represents the entire genome. Libraries may be either genomic DNA (both introns & exons are present) or cDNA (only exons are present).

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Ligation: The enzyme-catalyzed joining of two stretches of DNA or RNA into one.Northern blot: A method for transferring RNA from an agarose gel to a nitrocellulose filter, on which the RNA can be detected by a suitable probe.Oligonucleotide: A short, defined sequence of nucleotides (~2-200 long).Plasmid: A small, extrachromosomal, circular molecule of DNA that replicates independently of the host DNA.Polymerase chain reaction: An in invitro enzymatic method for the repeated copying (amplification) of DNAProbe: A molecule used to detect the presence of a specific segment of DNA or RNA. Common probes are cDNA molecules, synthetic oligonucleotides of defined sequence, or antibodies to specific proteins.Recombinant DNA: The altered DNA that results from the insertion of a sequence of deoxynucleotides that are not previously present.Restriction enzyme: An endonuclease that cleaves DNA at specific sites.Reverse transcription: RNA-directed synthesis of DNA, catalysed by reverse transcriptase.Southern blot: A method for transferring DNA from an agarose gel to nitrocellulose filter, on which the DNA can be detected but a suitable probe.Sticky-ended DNA: Complementary single strands of DNA that protrude from opposite ends of a DNA duplex.Tandem: Multiple copies of the same sequence that lie adjacent to one another.Transgenic: Introduction of new DNA into germ cells by its injection into the nucleus of the ovum.

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Any Questions?

“The important thing is not to stop questioning"

• Einstein

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كلمات باقية

إن كKKل مKKا يضKKايقني .. هKKو إنى ال أملك إال حياة واحدة أقدمها لبالدي

الزعيم الصيني

صان يات سن

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References:• Clinical Biochemistry: An illustrated colour text, A. Gaw et

al., Churchill Livingstone.

• Clinical Chemistry, W.J. Marshall, Mosby.

• Enzyme Tests in Diagnosis, D.W. Moss & S.B. Rosalki, Arnold.

• Clinical Chemistry: Interpretation and Techniques, A. Kaplan & L.L. Szabo, Lea & Febiger, Philadelphia.

• Fundamentals of Clinical Chemistry, N.W. Tietz, W.B., Saunders Company, Philadelphia, London, Tokyo ….

• Clinical Biochemistry, A.F. Smith, G.J. Beckett, S.W. Walker & P.W. H Rae, Blackwell Science Ltd, London…

• Interpretation of Diagnostic Tests, I. Wallach, Little, brown & Company, Boston, London …..

• Practical Clinical Biochemistry, H. Varley, A.H. Gowenlock & M. Bell, William Heinemann Medical Books LTD, London