Molecular diagnostics laboratory

2017

Contents

Exp. No. / Description / Page number
1 / Aseptic culture and safety roles / 3
2 / DNA isolation / 5
3 / RNA isolation / 9
4&5&6 / Human karyotype / 12
7 / Allele-specific PCR / 15
8 / Restriction fragment length polymorphism (RFLP) / 20
9&10 / Southern blot / 24
11 / Real time PCR / 28

Lab 1.

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 afreshpipet 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 tubescan 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 lidsclosedwhen you're not working with the samples.
Mediacan 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 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 themhot.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 spinALL 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 spinALL 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

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 acidis amoleculethat carries thegeneticinstructions used in the growth, development, functioning andreproductionof all known livingorganismsand manyviruses. 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, Vortex

Protein precipitation solution, restriction enzyme

Eppen dorf tube

Isopropanol, 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 solution
  6. 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.

  1. Spin 10 min at 13000-16000 rpm in a clinical centrifuge .
  2. 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
  3. 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 990microliters 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:

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

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 :

  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
  2. DPBS
  3. Trizol
  4. Chloroform
  5. 70% ethanol
  6. Blood sample
  7. 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;

it can also be held temporarily at -20 until permanent storage at -80. Repeated freezethaws

are to be avoided, so RNA should be aliquoted for transfer as soon as possible after

quantitation

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

Introduction

Akaryotypeis the number andappearanceofchromosomesin thenucleusof aeukaryoticcell. The term is also used for the complete set of chromosomes in aspeciesor in an individual organismand for a test that detects this complement or measures the number.

Karyotypes describe thechromosome count of an organismand what these chromosomes look like under a lightmicroscope. Attention is paid to their length, the position of thecentromeres, banding pattern, any differences between thesex chromosomes, and any other physical characteristics.The preparation and study of karyotypes is part ofcytogenetics.

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

The basic number of chromosomes in thesomaticcells of an individual or a species is called thesomatic numberand is designated2n. Thus, inhumans2n = 46. In thegerm-line(the sex cells) the chromosome number isn(humans: n = 23).

The study of karyotypes is important forcell biologyandgenetics, and the results may be used inmedicine. Karyotypes can be used for many purposes; such as to studychromosomal aberrations,cellularfunction,taxonomicrelationships, and to gather information about pastevolutionaryevents.

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 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.