Molecular diagnostics laboratory

2017

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Contents

Exp.

No.

Description Page

number1 Aseptic culture and safety roles 3

2 DNA isolation 5

3 RNA isolation 94&5&6 Human karyotype 12

7 Allele-specific PCR 158 Restriction fragment length polymorphism (RFLP) 20

9&10 Southern blot 24

11 Real time PCR 28

Lab 1.

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Aseptic technique and safety roles

Aseptic technique

Aseptic technique refers to laboratory practices to avoid exposing preparations to bacteria, mold, and other contaminants. We apply aseptic technique in a conventional laboratory environment when working with bacterial plates, DNA or protein preparations, etc. Materials are frequently sterilized before use, but sterile conditions are not necessarily maintained during use. The phrase "sterile technique" refers to more stringent practices to prevent the slightest contamination whatsoever. Surgeons apply sterile technique, as to researchers who culture cells and tissues.

General Guidelines

1. Maintain a clean work area

2. Use a fresh pipet tip for every transfer (tips should be DNase/RNase free)

3. Wear gloves to prevent contamination (of yourself as well as your experiment)

4. Sterilize solids and liquids by autoclaving 20 minutes at 121°C at 15 psi

5. Read carefully the instruments before use any reagent , because some reagent is carcinogen and some is sensitive for light .etc.

Microcentrifuge/conical centrifuge tubes can easily be contaminated by contact with non-sterile surfaces (e.g., your fingers) or by air borne particles. Be careful when transferring solutions from one tube to another. Also, keep the lids closed when you're not working with the samples.

Media can be also contaminated by contact with non-sterile surfaces or by air borne organisms. Remove lids and coverings carefully avoiding contact with any part of the cover that may contact the media; minimize the amount of time the container is exposed to air. Lids and coverings should be held with media side down at all times. Air borne contaminants are usually falling downward. Replace the coverings carefully so that the rim of the container makes contact only with sterile surface of the inside of the cap.

The use of a flame helps maintain aseptic materials. Working near a flame can decrease air borne contamination. The flame is also used to singe surfaces to maintain sterility. The mouth of the tube or flasks is passed through the flame before and after pouring. The cap or cover is

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also passed through the flame prior to replacing on the container.

Caution: The flame is used to singe the surfaces only. Do not hold the items in the flame to make them hot.Glass flasks, even Pyrex, can break from the heat or when the cooler media hits the hot surface.

Notes on Molecular Biological Procedures

Centrifugation

1. DO NOT PUT TAPE ON TUBES!

2. ALWAYS balance the load in the centrifuge

3. Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the rotors

4. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the micro-centrifuge hold the "SHORT" key for about 5-10 seconds

5. DO NOT SLAM THE LIDS! (this action breaks the latch mechanisms)

Pipetting Small Volumes

1. Before beginning the procedure, thaw all frozen reagents and mix well

2. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube as described above

3. Touch only the very tip to the surface of the solution (i.e., do NOT submerge the pipet tip into the solution)

4. Most enzyme stocks are in 50% glycerol; these solutions are quite viscous and liquid will stick to the outside of the pipet tip so touch only the surface

Lab 2. DNA isolation

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Objectives: 1. To compare between prokaryotic and eukaryotic DNA.2. To use centrifuge for separation technique 3. To be familiar with gel electrophoresis .4. Compare the integrity of eukaryotic and prokaryotic DNA

Introduction:DNA Deoxyribonucleic acid is a molecule that carries the genetic instructions used in

the growth, development, functioning and reproduction of all known living organisms and many viruses. DNA is a nucleic acid, made of carbon, hydrogen, oxygen, nitrogen, and phosphorous. DNA can be considered the hereditary " code of life" because it possesses the information that determines an organism's characteristics and is transmitted from one generation to the next. You receive half of your genes from your mother and half from your father. The more closely related organisms are, the more similar their DNA.

DNA is in almost every prokaryotic and eukaryotic cells. In your body, the length of DNA per cell is about 100,000 times as long as the cell itself. However, DNA only takes up about 10% of the cell's volume. This is because DNA is specially packaging through a series of events to fit easily in the cell's nucleus. The structure of DNA, the double helix, is wrapped around protein, folded back onto itself, and into a compact chromosome.

Bacterial DNA differs from that of eukaryotic cells: each bacterial cell has a single circular double helical DNA molecule. In addition it has several copies of very smaller circular DNA molecules called plasmids.

Materials:Human blood sample, bacterial sample Red cell lyses buffer and white cell lyses buffer, tissue and cell lyses solution.Clinical centrifuge, VortexProtein precipitation solution, restriction enzymeEppen dorf tubeIsopropanol, 70% ethanol, loading buffer

Procedure: I- Isolation of eukaryotic DNA (e.g human blood): 1. Add 900 Ml of Red cell lyses buffer to 600 Ml of blood sample and mix completely by

vortex.2. Incubate the solution for 15 min at room temperature.3. Spin 10 min at 13000-16000 rpm in a clinical centrifuge.4. Discard supernatant and add 300 Ml of white cell lyses buffer to the pellet.5. Add 100 Ml of protein precipitation solution to the solution6. Vortex for 2 min and centrifuge for 10 min at 13000-16000 rpm.Take supernatant to a newly Eppen dorf tube and add 300 Ml of isopropanol and completely mix by inverting.7. Spin 10 min at 13000-16000 rpm in a clinical centrifuge .

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8. Discard supernatant and wash the pellet with 300 Ml of 70% ethanol; air dry for 20 min and resuspend in 70 Ml of rehydration solution

9. Leave overnight at 4⁰C to resuspend completely.

Estimation of quantity and quality of isolated DNA

Methods for Determining DNA Yield and Purity:• DNA quantity and quality can be assessed using several different methods

include:1- Absorbance by spectrophotometer or Nanophotometer.2- Agarose gel electrophoresis .

• The most common technique to determine DNA yield and purity is also the easiest method—Absorbance

• Nucleic acids absorb light at 260 nm ,the A260 reading should be between 0.1–1.0. However, DNA is not the only molecule that can absorb light at 260nm. Since RNA also has a great absorbance at 260nm will contribute to the total measurement at 260nm.

Quantification of DNA by spectrophotometry:• Using TE buffer as the diluent, make an appropriate dilution of your DNA depending on

the size of the cuvettes available (e.g. for 1ml cuvettes, dilute 10 microliter DNA solution in 990 micro liters of TE).

• Determine the absorbance of DNA at 260 using TE as the reference solution (i.e. as a blank)

• Using a conversion factor :• one optical density unit (or absorbance unit) at 260 nm is equivalent to 50

microgram/mL of DNA and 40microgram/mL of RNA. • Multiply the absorbance reading by the conversion factor and the dilution factor to find

the concentration of nucleic acid.

• Pure DNA Concentration (microg/ml) = (A260 reading – A320 reading) x dilution factor x 50microg/ml

• Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

• DNA Yield (microgram) = DNA Concentration x Total Sample Volume (ml)

• A reading at 320nm will indicate if there is turbidity in the solution, The 320 nm absorbance is used to correct background absorbance. (BLANK)

• Example 1. A DNA preparation diluted 1:100 yields an absorbance reading of 0.200 at 260 nm.

• To obtain the concentration in micro gram/mL:

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0.200 absorbance units x 50 microg/mL x 100 =1000 microg/mL

• The yield of the sample is calculated using the volume of the preparation.

• If in the case illustrated above, the DNA was eluted or resuspended in a volume of 0.5 mL

• The yield would be: 1000 microg/mL x 0.5 mL =500 microgram

Quality of DNA using spectrophotometer:

– DNA UV absorbance at 260 nm.– protein UV absorbance at 280 nm .– The ratio of the absorbance at 260 nm/280 nm is a measure of the purity of a

DNA sample from protein contamination; it should be between 1.7 and 2.0

• The absorbance of the nucleic acid at 260 nm should be 1.7–2.00 times more than the absorbance at 280 nm.

• If the 260 nm/280 nm ratio is less than 1.7, the nucleic acid preparation may be contaminated with unacceptable amounts of protein and not of sufficient purity for use.

• Such a sample can be improved by reprecipitating the nucleic acid step of the isolation procedure

• DNA Purity (A260/A280) = (A260 reading – A320 reading) \• (A280 reading – A320 reading)

• A DNA preparation with a ratio higher than 2.0 may be contaminated with RNA.

• If RNA may interfere or react with DNA detection components, RNase should be used to remove the contaminating RNA.

• The ratio of the absorbance at 260 nm/230 nm is a measure of the purity of a DNA sample from organics and/or salts; it should be about 2.0.

• Low A260/A230 ratio indicates contamination by organics and/or salts

Some notes about Nanophotometer:

• Don’t need dilution• The volume required for measurement 3-5 microliters• The concentration given in nanogram \microliters

Quality from Agarose Gel Electrophoresis:

• Quality of DNA extracted is assessed using the following simple protocol:• Mix 5 µL of DNA with 5 µL of loading Dye• Load this mixture into a 1% agarose gel• Stain with ethidium bromide • Electrophorese at 70–80 volts, 45–90 minutes

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DNA Quality from Agarose Gel Electrophoresis

• High molecular weight band (>48.5 kb) • Smearing indicates DNA degradation (or too much DNA loaded).

Troubleshooting Nucleic Acid Preparation Methods

• Problem: No or low nucleic acid yield.– Make sure that ample time was allowed for resuspension or rehydration of

sample.– Repeat isolation from any remaining original sample .– Concentrate dilute nucleic acid using ethanol precipitation

1-The blood sample may contain too few white blood cells. Draw new blood samples.

2- The white blood cell pellet was not resuspended thoroughly .

3- The blood sample was too old. Best yields are obtained with fresh blood. Samples that have been stored at 2–8°C for more than 5 days may give reduced yields.

4- The DNA pellet was lost during isopropanol precipitation. Use extreme care when removing the isopropanol to avoid losing the pellet.

• Problem: Poor nucleic acid quality– If sample is degraded, repeat isolation from remaining original sample, if

possible.• If sample is contaminated with proteins or other substances, clean it up by re-isolating

repeating the protein removal step of the isolation procedure.

Lab 3. RNA isolation

Objectives :

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1. To be familiar with RNA extraction by manual method and kit method 2. To use centrifuge for separation technique 3. To be familiar with gel electrophoresis

Introduction ;

Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation, and expression of genes. RNA and DNA are nucleic acids, and, along with proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the letters G, U, A, and C to denote the nitrogenous bases guanine, uracil, adenine, and cytosine) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function where RNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) then links amino acids together to form proteins.

Note:

RNA is very easily degraded by ever-present RNAses. Therefore, all of the tubes and solutions in this protocol must be RNAse-free (autoclaving does NOT inactivate RNAses). One cannot overemphasize the need for a clean work environment when working with RNA.

Material;

1. RBC ,S lysis buffer

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2. DPBS3. Trizol4. Chloroform5. 70% ethanol6. Blood sample7. Sterile apendrof tube 8. Sterile distilled water

Procedure:

1) Transfer contents of tube into a 50 ml polypropylene conical centrifuge tube. 2) Bring volume to 45 ml with RBC Lysis Buffer (recipe follows protocol). 3) Let stand at room temperature for 10 minutes. 4) Pellet cells at 600 x g (approx 1,400 rpm) for 10 minutes in a room temp centrifuge 5) Carefully decant supernatant. 6) Gently resuspend the pellet in 1 ml of RBC Lysis Buffer and transfer to a 1.5 ml

microcentrifuge tube. – Let stand for 5 minutes. 7) Pellet cells for 2 minutes by centrifuging in a microfuge at room temperature at 3000 rpm. 8) Carefully aspirate the supernatant. 9) Resuspend the pellet in 1 ml of sterile DPBS. 10) Pellet cells as in step 7. 11) Carefully aspirate the supernatant. 12)Add 1200 μl of TRIzol solution to each tube and resuspend the cells. Note: for a full 8 ml

blood tube, the 1200 ul TRIzol solution can be split into 2, 600 μl aliquots and frozen at -80 C until further processing.

13)Add 0.2 ml of Chloroform (CHCl3) and vortex each tube for 15 seconds, ONE AT A TIME.

14) Centrifuge the samples at 13,000 rpm for 10 minutes at 4°C. 15) Remove the upper phase and transfer to a clean microcentrifuge tube. Be careful not to

remove any of the white interface when collecting the upper phase of the extraction 16) For the future collection of micro RNA (miRNA), carefully remove ~20% of the volume

of the upper phase from step 16 and place into another clean, labeled, 1.5ml microfuge tube. Store this aliquot at -80 C until further processing.

17) To the remaining upper phase from step 16, add an equal volume of cold isopropanol and invert to mix.

18) The samples can be placed in a -20°C freezer to precipitate. 19) Samples are centrifuged at 13,000 rpm for 10 minutes at 4°C. Note: you may be able to

see a small white pellet of RNA at the bottom of the tube after this step. 20) Carefully decant the supernatant, and rinse the pellet with 0.5 ml of ice-cold 75%

ethanol. The 75% EtOH should be prepared RNase-free and stored at -20 C. 21) Centrifuge the samples at 13,000 rpm for 10 minutes at 4°C. 22)Decant the supernatant. 23)Using a pipettor, carefully remove all of the remaining liquid in the bottom of the tube 24)Allow the pellet to dry for 5 to 10 minutes to remove any remaining ethanol.25)Dissolve the RNA pellet by adding 20 μl of RNAse-free H2O to each sample.26) RNA should be quantitated within 2 hours of elution. It can be kept at 4 C until that time;

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it can also be held temporarily at -20 until permanent storage at -80. Repeated freezethawsare to be avoided, so RNA should be aliquoted for transfer as soon as possible afterquantitation

Lab 4&5&6

Human karyotype

Objectives:1. Students will be able to demonstrate a micro-technique for reliable chromosomal

analysis of leucocytes obtained from peripheral blood.2. Students will be able to prepare a karyotype from the chromosomes of a normal human

male or female.

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3. Students will be able to use the karyotyping techniques for diagnosing a chromosomal disorder.

Introduction A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number.

Karyotypes describe the chromosome count of an organism and what these chromosomes look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics.

The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size.

The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Thus, in humans 2n = 46. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23).

The study of karyotypes is important for cell biology and genetics, and the results may be used in medicine. Karyotypes can be used for many purposes; such as to study chromosomal aberrations, cellular function, taxonomic relationships, and to gather information about past evolutionary events.

Chromosomes of similar size and morphology are grouped together by letter; the chromosomes can be arranged in 7 groups (A, B, C, D, E, F, G). thus group A contains pairs #1, #2, and #3

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Karyogram of human male using Gimsa stain

chromosomes (look to the next figure). Karyotyping is a technique that allows for visualization and identification of human metaphase chromosomes.

This technique can be used to assay the normalcy of an individual's chromosomes and to assay for various genetic diseases such as Down's syndrome and Klinefelter's syndrome…etc.

To create a karyotype, chromosomes from cell are arranged, stained and photographed. The photograph is enlarged and cut up into individual chromosomes. The homologous chromosomes can be distinguished by length and by the position of the centromere.

A chromosome is divided by its centromere into short arm (p) and long arm (q) chromosomes can be classified by the position of their centromere:

Metacentric: if its two arms are equal in length. Submetacentric: if arm's lengths are unequal. Acrocentric: if the q arm is so short that is hard to observe, but still present.

A blood sample is taken and white blood cells grown in special medium for three days under the influence of the mitotic stimulant ( phytohemagglutinin "PHA") to enter into mitosis by DNA replication. After 68-72 hours, a mitotic inhibitor (cholchicin) is added to the culture to stop mitosis in the metaphase stage. After treatment by hypotonic solution(KCl ) to causes a swelling of the cells and allow dispersion of the chromosomes within the cell membrane.

Fixation is used as a washing solution to lyse the remaining red blood cells and remove come chromosome's protein. After staining, chromosomes can be microscopically observed and evaluated for abnormalities.

Materials : Heparinized whole blood. Heparin sodium injection. Peripheral blood karyotyping medium with PHA (RPMI 1640) Incubator 5% CO2 at 37°C 0.075 M KCl Fixative solution (3x methanol : 1x glacial acetic acid ) Giemsa stain solution Slides and microscope

Procedure:1. Inoculate 0.5ml of heparinized whole blood into tube with 10ml of karyotyping

medium.2. Inoculate the tubes in incubator with 5% CO2 at 37°C for total of 72 hours.3. After total of 69 hours from seeding add 100µl of colcemid solution to each culture

tubes.4. Incubate the tubes at 37°C for an additional 20-30 min.

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5. Spin at 500g (1500 rpm) for 7 min.6. Remove the supernatant and re-suspend the cells in 5ml of hypotonic solution 0.075

KCl pre-warmed to 37°C.7. Incubate at 37°C for 15 min.8. Add drop by drop (with vortexing) 1ml fresh ice cold fixative.9. Spin at 500g (1500 rpm) for 7 min.10. Remove the supernatant, agitate the cellular sediment and add drop by drop (with

continuous vortexing), 5ml of fresh, ice cold fixative.11. Leave at 4°C for 20 min.12. Repeat steps 9 and 10, until the supernatant is clear.13. Spine at 500g (1500 rpm) for 7 min.14. Re-suspend the cell pellet with a 1.5ml of fresh fixative.15. Drop 4-5 drops, from a high of approximately 30 cm onto a clean slide and blow

carefully on the drops for spreading them on the slide.16. Put the slide on a 45°C heated plate for 2-4 min.17. Heat the slide to 60°C for overnight or to 90°C for 90 min.18. Place the slides and flood them Gimsa stain solution for 8 min.19. Gently rinse the slide in distilled water and air dry.20. Observe the chromosome under microscope by using 10, 40 and 100x and photograph

it and cut each chromosome from the photograph and arrange the chromosome according to the size and position of centromere.

Lab 7

Allele-specific PCR

Objectives:

1. To be familiar with allele-specific PCR 2. To use allele specific PCR in diagnosis of disease

Introduction :

Allele-specific polymerase chain reaction (ASPCR) is an application of the polymerase chain reaction (PCR) that permits the direct detection of any point mutation in human DNA by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. ASPCR works because an oligonucleotide primer that forms a 3 mismatch with the DNA ′template will be refractory to primer extension by Thermus aquaticus DNA polymerase. Therefore, oligonucleotide primers specific for all known alleles can be synthesized and used to

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detect the alleles in DNAs of unknown genotype. ASPCR has already been used in DNA-based diagnostic techniques involving the diagnosis of genetic and infectious diseases. For example , the use of ASPCR for the direct diagnosis of sickle cell anemia and discuss different mythological aspects of ASPCR.

Allele-specific polymerase chain reaction (AS-PCR), also known as amplification refractory mutation system (ARMS) or PCR amplification of specific alleles (PASA) is a PCR-based method which can be employed to detect the known SNPs.

In this approach, the specific primers are designed to permit amplification by DNA polymerase only if the nucleotide at the 3’-end of the primer perfectly complements the base at the variant or wild-type sequences. After the PCR and electrophoresis, the patterns of specific PCR products permit the differentiation of the SNPs.

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PCR technique is used to have a large quantity of DNA to perform allele specific

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy and cheap tool to amplify a focused segment of DNA, useful for such purposes as the diagnosis and monitoring of genetic diseases, identification of criminals (in the field of forensics), and studying the function of a targeted segment of DNA.

he method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. PCR is not generally considered to be a recombinant DNA method, as it does not involve cutting and pasting DNA, only amplification of existing sequences.

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Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.

In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

A basic PCR set up requires several components and reagents.[9] These components include:

DNA template that contains the DNA region (target) to amplify Taq polymerase, a DNA polymerase that is heat resistant,[10] so that it can remain

intact during the DNA denaturation process. Two primers that are complementary to the 3' (three prime) ends of each of

the sense and anti-sense strand of the DNA target. Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide

triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.

Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

Bivalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[11]

Monovalent cation potassium ions

Steps of PCR :

Initialization step (Only required for DNA polymerases that require heat activation by hot-start PCR): This step consists of heating the reaction to a temperature of 94–96 °C , which is held for 1–9 minutes.

Denaturation step : This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the

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DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind.

Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,[14][15] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction,

Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

Material:

1. DNA samples 2. Sterile tips 3. Micropipte 4. Sterile water5. Common primer 6. Normal primer 7. Mutant primer 8. Master mix 9. Sterile PCR tubes 10. Agarose powder 11. Electrophoresis device

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Procedure :

For each DNA sample , design 2 PCR tubes , one for normal primer and the second for mutant primer .

A. First tube1. Add 2µl of common primer 2. Add 2 µl of normal primer3. Add 4 µl of sterile D.W 4. Add 10 µl of master mix5. Add 2 µl of DNA sample B. Second tubes , as mentioned in first tube , but instead of normal primer ,add mutant

primer .

Lab 8

Restriction fragment length polymorphism (RFLP)

Objectives:

1. Define restriction fragment polymorphism 2. To know the type of polymorphism3. To be familiar with PCR technique 4. To be familiar with diagnosis by using RFLP technique

Introduction:

A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1–2% of a population

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Polymorphisms may or may not have phenotypic effects.

Types of Polymorphic DNA Sequences:

1. VNTR: variable number tandem repeats (8 to >50 base pairs)2. STR: short tandem repeats (1–8 base pairs)3. SNP: single-nucleotide polymorphisms

Difference in DNA sequence can be detected by RFLP

RFLP : is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules from differing locations of restriction enzyme sites, In RFLP analysis, the DNA sample is broken into pieces and (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis, and transferred to a membrane via the Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe, we will say about southern blot in the next lab .

An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis. It requires a large amount of sample DNA.

RFLP analysis was an important tool in

1. genome mapping, 2. localization of genes for genetic disorders, 3. determination of risk for disease, 4. paternity testing.5. crime scenes

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PCR technique is used to have a large quantity of DNA to perform RFLP

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Material:1. sterile tips

2. micropipte (1000 µl,50,10)

3. sterile water

4. PCR tubes

5. sterile micro-centrifuge tube

6. DNA samples

7. restriction enzyme

8. thermal cycler

9. agarose powder

10. balance

11. electrophoresis devise

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Procedure:

For each PCR tube,add

1. 10µl master mix

2. 4 µl water

3. 2 µl forward primer

4. 2 µl reverse primer

5. 2 µl DNA sample

Put the tubes in thermal cycler , after the reaction is done , add 2 µl of restriction enzyme to PCR tube and leave it overnight , in the next day , prepare 3% agarose gel and load the sample (4µl) in the wells , and allow the samples to run during electrophoresis .

Read the bands under UV device

Lab 9&10

Southern blot

Objectives :

1. To be familiar with southern blot technique 2. To learn about how southern blot is used in diagnosis

Introduction :

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples, the DNA detected can be single gene or it can be a part of large piece of DNA such as viral genome . Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar

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principles, but using RNA or protein, have later been named in reference to Edwin Southern's name.

Southern blot can be used as later step of RFLP to determine the length of fragments .

Principle:

The key to this method is hybridization

Hybridization :it is the process of forming double stranded DNA molecule between a single-stranded DNA probe and single-stranded target DNA

There are two important feature of hybridization :

1. The reaction is specific ,the probe will only binds to target with complementary sequence .

2. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules .

Summary of procedure :

1. Extract and purify DNA from the cells 2. DNA is restricted with enzyme 3. Separated with electrophoresis 4. Denature DNA 5. Transfer to nitrocellulose or nylon membrane6. Add labeled probe for hybridization 7. Wash off un bounded probe8. Autoradiograph

Application:

1. to identify specific DNA in DNA sample 2. to isolate desired DNA for construction of rDNA3. in RFLP4. in DNA fingerprinting5. identify mutation and deletion6. used in prognosis of cancer 7. diagnosis in HIV-1 and infectious disease

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Materzial :

1.sterile tips

2.micropipte

3.agarose gel

4.balance

5. electrophoresis device

6.nitrocellulose or nylon membrane

7.tisssue paper, filter paper

8. 20x ssc

9.DNA samples

10.probes

11.restriction enzyme

Procedure:

1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.

2. The DNA fragments are then electrophoresed on an agarose gel to separate them by size.

3. If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl. This depurinates the DNA fragments, breaking the DNA into smaller pieces, thereby allowing more efficient transfer from the gel to membrane.

4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The

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denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later hybridization to the probe, and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [

5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction, 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel onto the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.

6. The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.

7. The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labeled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non-specific binding of the probe.

8. After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.

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Lab 11

Real time PCR

Objectives:

1. To be familiar with real time PCR device 2. To comparison between conventional PCR and real time PCR

Introduction :

A real-time polymerase chain reaction (qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA molecules (semi quantitative real-time PCR).

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Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence.

Basic principles

Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.

The PCR process generally consists of a series of temperature changes that are repeated 25 – 50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain; the second, at a temperature of around 50-60 °C, allows the binding of the primers with the DNA template;] the third, at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to increase their number during the change between the alignment stage and the denaturing stage. In addition, in four steps PCR the fluorescence is measured during short temperature phase lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used. The temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the bonding temperature of the primers.

Chemical classificationReal-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes.

Non-specific detection: Real-time PCR with double-stranded DNA-binding dyes as reportersA DNA-binding dye 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 measured at each cycle. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can potentially interfere with, or prevent, accurate monitoring of the intended target sequence.

In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of fluorescent dsDNA dye. Then the reaction is run in a real-time PCR instrument, and

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after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). This method has the advantage of only needing a pair of primers to carry out the amplification, which keeps costs down; however, only one target sequence can be monitored in a tube.

Specific detection: fluorescent reporter probe method

Fluorescent reporter probes detect only the DNA containing the sequence complementary to the probe; therefore, use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.

The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.

1. The PCR is prepared as usual (see PCR), and the reporter probe is added.

2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.

3. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.

4. Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to exponential increase of the product is used to determine the quantification cycle (Cq) in each reaction.

Fusion temperature analysisReal-time PCR permits the identification of specific, amplified DNA fragments using analysis of their melting temperature (also called Tmvalue, from melting temperature). The method

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used is usually PCR with double-stranded DNA-binding dyes as reporters and the dye used is usually SYBR Green. The DNA melting temperature is specific to the amplified fragment. The results of this technique are obtained by comparing the dissociation curves of the analysed DNA samples.[11]

Unlike conventional PCR, this method avoids the previous use of electrophoresis techniques to demonstrate the results of all the samples. This is because, despite being a kinetic technique, quantitative PCR is usually evaluated at a distinct end point. The technique therefore usually provides more rapid results and / or uses fewer reactants than electrophoresis. If subsequent electrophoresis is required it is only necessary to test those samples that real time PCR has shown to be doubtful and / or to ratify the results for samples that have tested positive for a specific determinant

Advantages over PCR

Real time PCR has many advantages over normal PCR

1. It doesn’t require gel preparation like traditional PCR 2. It is not time consuming like normal PCR

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3. Less complexity at the quantification of samples

Applications[

There are numerous applications for quantitative polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and basic research. Uses of the technique in industry include the quantification of microbial load in foods or on vegetable matter, the detection of GMOs (Genetically modified organisms) and the quantification and genotyping of human viral pathogens.

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