Laboratory Medicine

Assay development for superior detection of sex chromosome aneuploidies And Copy number variation and disease

By Alamri, Sultan Saad School of medical science RMIT University

DUE DATE: 14th Nov 2011

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Declaration

I declare that this thesis contains my original work.

Information from published or

unpublished sources has been clearly acknowledged within the thesis. None of the work contained in this thesis has been submitted to qualify for any other academic award. The content of the thesis is a result of the work, which has been carried out during the enrolled period of the program.

Sultan Alamri 14th Nov 2011

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Acknowledgements

My research would have not materialised if I did not have the support, and encouragement from a number of people.

First and foremost, I would like to thank my government, Associate Ministry of higher education, for their invaluable guidance and steadfast encouragement from the time I started as master student in RMIT and for providing me with scholarships to pursue my studies. I have enjoyed working with you and hope to have the opportunity to do so again.

I would like to express my sincere thanks to Associate Professor Peter Roche and Dr. David Godler for their encouragement and for giving so generously of their time and expertise.

I am thankful to Murdoch Childrens Research institute for providing me with materials to pursue my project. I am deeply indebted to my family for giving me a supports during my master studies.

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Thesis abstract:

The aim of this study was to evaluate the specificity and sensitivity of the real time PCR method for sex determination and chromosome anaiploidies. The human sex determining region on the Y chromosome has been identified and the gene has been named as SRY gene. The normal function of SRY gene is to provide instructions to make a particular protein which called the sex determining region Y protein. This protein binds to specific region of DNA and control the activity of gene.SRY protein causes a fetus to develop as male. Normal people have 46 chromosomes; two of the 46 chromosome are called sex chromosome, known as X and Y. sex chromosome determine whether a fetus will develop female or male characteristics. Female have XX and male have XY.

DNA samples were extracted from blood and saliva specimens subjected to real time PCR. Amplification of SRY as a Y chromosome marker and globin as an interval control (2copies). The DNA test involves the amplification of specific region of SRY gene. The present of SRY amplified product directly indicates the presence of Y chromosome. Also, if there is an extra copy from Y chromosome as in some sex-linked disorders, the amplification showing double intensity as compared to the normal male control. Otherwise, it shows 0 intensity as in female on turners sample (X) or present same amount of intensity in normal male sample. For the real time you need to setup standard curve as mention before to measure the value of unknown concentration. The PCR cycle at which fluorescence exceeds background and a significant increase in fluorescence is observed is the threshold cycle. The Ct value is correlated with the starting template amount since it corresponded to PCR product accumulation. The quantification of the initial copy numbers of the unknown samples are facilitated by comparing the Ct values of the unknown samples to the standard curve.

This project looks at the discovery and assessment of the clinical significant of copy number variation detected in patients with a wide range of disorders, including mental retardation, epilepsy, autism, developmental and dysmorphism.

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My role in this project was to setup master mix and run gel for 2000 PCR products to identify the genotype wither heterozygote or homozygote. Heterozygous (+/-) means that segment of DNA having two different alleles. So that mean, each gene has two different copies; one is inherited from the father and the other from mother. When PCR products run on gel electrophoresis, bands were observed by using UV light. However, possessing two identical alleles means homozygous (+/+).

Genotyping identification assay is mainly based on PCR technology whether traditional or real time PCR. Murduch children research institute (MCRI) uses the traditional PCR that involves the using of UV light. This technique is not automated and requires post PCR processing (gel electrophoresis); therefore, it is time consuming. Moreover, the results are based on size discrimination. However, for each and every PCR application, optimal reaction conditions need to be ensured for the successful amplification of the PCR product. The basic reaction mixture consists of all the components necessary to make new segment of DNA in the PCR process template DNA, primers (forward and reverse), reaction buffer; magnesium chloride (MgCl2), dNTPs and aheat stable DNA polymerase.

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Table of contents: Declaration. Acknowledgement. Thesis abstract... Part one: introduction... Cell cultures Lymphoblast cultures Traditional PCR Copy number variation and disease ... Optimization the condition for the real time PCR. DNA extraction.. DNA methylation... Fragile X. DNA microarray

Part two: introduction... Assay development for superior detection of sex chromosome aneupluidies.. BACs-on Beads assay... References..

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Introduction: Victorian clinical genetics services pathology is part of Murdoch children research institute. VCGS provide both, genetics testing and genetics counseling and it is provided by scientists and genetists consulting. It also has research groups focusing on developing new diagnostic assays.

Laboratories within the VCGS consist of molecular genetics laboratories, cytogentics laboratories, maternal screening, prenatal serum screening laboratories and biochemical testing laboratories.

I have been located in cytogenetic research group that providing cytogentics screen to examine the chromosome abnormality such as deletions, duplications or inversion in clinical specimens.

The cytogentics laboratory is subdivided into a different sections or departments that include the prenatal , oncology and the blood DNA section.

The responsibility of DNA section is to extract the DNA from clinical samples such as tissue, blood, amniotic fluids, or saliva. The DNA section is also involved for performing different techniques to identify the mutations in samples.

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The blood department has responsible for processing the samples that include patient data entry, giving a numbers for each sample in lab. Also this department involve into the preparation of slides for chromosomal banding.

Department of oncology is accountable for

preparation of slides and examining for any

chromosomal abnormalities that are known to cause cancer or sex linked disorders. One such example is that TEL-AML translocation which is lymphoblastic leukemia. chromosomal abnormality in acute

Department of prenatal is responsible for processing the pre-natal specimens and performing of pre-natal testing.

Safety and regulatory requirements: All students who are about to start doing their project in this institute (VCGS) are required to do safety session before they starting of their work. This session is called occupational health and safety to provide the principle of dealing with different lab equipment and chemical detergents that is used in VCGS laboratories.

There are some basic roles and safety procedures that have been applied all time and must be followed by staff and student that are working inside the laboratories are included:

Disposal of waste must be done according to the laboratory procedures. Closed shoes must be worn all time. No food or drink inside laboratories. Long hair must be tied. Horseplay and practical jokes must be avoided. Gloves and protective equipment must be worn when handling infectious materials. All cuts must be covered before starting any work. Spills must be cleaned up immediately and all incidents must be reported following the lab reporting procedures.
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Everyone in the lab must be able to identify the hazardous substances and be able to handle them according to the lab procedures.

In the end of this week we have learnt how to deal with chemical spill and basic safety roles .Also, there was no procedure undertaken by me because it was my first week of orientation and registration. My work experience is involved with the cytogentics research module under the supervision of Dr.David. Doctor David main research focus is on development of new methods to detect the abnormality of chromosome. For now, I am involved in a project with one of the PhD student, Devika.

Cell cultures: Cell culture is referred to the removal of cells from tissue and following growth under controlled conditions. Those cells could be directly removed from tissue and separated by using enzymatic before cultivation or it can be cultured from cell line that has already been obtained. Cell cultures are one of the main tools that used in molecular lab, providing excellent cell modal for studying normal physiology and mutagenesis of cells. Also the main advantage of this technique is that the consistency and reproducibility of results can be obtained from batch of clonal cell. Lymphoblast cultures: Lymphoblast cell line is obtained from the patient lymphocytes using EVB transformation. All cells must be free of bacterial and fungal contamination. The basic condition for lymphoblast culture Medium: RPMI , 20% FCS, L-glutamine Culture conditions: T25 tissue culture dishes with 20 ml medium, 37 C under CO2

Successful cell culture depends on many factors including the quality of cells and reagents that you used, sterile technique, the amount of the experience you have and the right laboratory equipments. Proper setup is important and having everything you need close by.

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The basic equipments you find in lab tissue culture lab including the cell culture hood, incubator, water bath, a centrifuge fridge freezer, microscope and heamocytometer. Also, you need cell culture flask medium and anther reagents (ethanol...etc), pipette and waste container. The hood provides sterile environment to work with your cells without contamination. Always open cell culture dishes and reagents bottle in sterile environment. Outside the hood you always keep pipette nearby. Pipette replaced in hood during use and only opens inside the sterile field. Cells can be counted manually using hemocytometer. Incubator provides appropriate environment for cell growth. Carbon dioxide and temperature setting depends on cell type and medium selection. Mammalian cells are cultured at 37C in the presence of 57% of carbon dioxide with high humidity. Inside the incubator keep flask lids loosely to ensure that the air inside more freely to maintain gases exchange. Finally, view every culture under the microscope daily to check for cell heath and growth and ensure of contamination. The shape and appearance of culturing lymphoblast cells are spherical in shape.

Common problems in growing lymphoblast: Many factors can be affected the growth of lymphoblast including: pH, temperature, long of time in continuous culture, contamination and depletion of L-glutamine. To be maintained these factors, store all medium and reagents according into the instruction on the label. Some component will degrade when left at room temperature or expose to light for prolonged time. Medium is pre-warmed to room temperature before addition to cell and added L-glutamine.

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Traditional PCR Polymerase chain reaction or PCR is alternative to clone for isolated DNA sequence from biological samples. Also, its enables researchers to produce a huge number of copies of target any specific nucleic acid sequence in two to three hours. The procedure has wide application in diagnostic to determine wither the clinical samples contains a microbial pathogens nucleic acid sequence or the laboratory scientist may use the PCR to amplify large quantities of desired sequence that present in complex mixture of other DNA molecules for research purpose. To perform the PCR you must know the sequence of nucleic acid that you want to amplify. For each and every PCR application, optimal reaction conditions need to be ensured for the successful amplification of the PCR product. The basic reaction mixture consists of all the components necessary to make new segment of DNA in the PCR process template DNA,both primers forward and reverse , reaction buffer; magnesium chloride (MgCl2), dNTPs and aheat stable DNA polymerase. The Master Mix reagents include:

Final concentration

Component

Function

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water 10x Buffer maintains the reaction at the proper pH

0.20mM

dNTPs

Provides nucleosides for the synthesis of DNA

1U

Hot Start Taq

A heat stable enzyme that adds DNTPs to the DNA

1u

Template DNA

The interest DNA will be amplified.

0.2uM

Primer

Short segment of DNA (20bp) that binds to DNA template and allowing enzyme to initiate the reaction or to get the enzyme started, and a DNA template to copy it.

However, there are three main steps identified by incubating the sample in different temperature, and those steps make up which called PCR cycles as shown in the figure below. 1- Denaturation 2- Primer annealing to template DNA 3- Primer extension

Briefly, the Polymerase chain reaction started with denaturation step. Sample is heated to around 95C for 15 min to separate the double strand of the DNA. The temperature is dropped to 60C for 30 sec. This step allows the primers to anneal to complementary sequences. The temperature is increased to 72C to allow the Taq polymerase to bind to the primers and extend or synthesize the new strand and this process is called cycle one. The process of denaturation, annealing and extending are repeated to make a new DNA copies.

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Figure 1. PCR cycle. The three different temperatures which make up a single cycle. The DNA denaturation section, primer annealing section and the primer extension.

The simplest way to detect if there is any product is to load some sample that taken from the PCR product, along with marker, into an agarose gel which contains ethidium bromide. DNA bands can then be visualized under UV. By comparing product bands with bands from markers, you should be able to estimate the size of any fragments.

Common problems in setting master mix for PCR:

During this week, I encountered in some problems while preparing trial master mix for the traditional PCR. Accurate pipetting of small amount is very critical to achieve a successful PCR. In cytogenetics lab they use different type of pipette such as multi-channel pipette, electronic pipette etc. So to solve this kind of problem you need to be skillful in pipetting.

Also, some of those pipettes seem to be old and need to be recalibrated as result of faults reading. Pipette calibration is important for accurate pipetting.

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During the set up master mix for PCR, I noticed that enzymes tube was out of the freezer for long time and I got no bands when I run the PCR product on gel. So, to avoid this kind of mistake you need to make sure that all reagents are kept on proper condition and the components for the master mix on ice bag during the procedure. In this case, I repeated the same procedure but using fresh reagents to avoid the previous mistake.

I have participated in study done at the VCGS using traditional PCR machine and the aim of the study was to screen the genotype for 100 DNA samples using different primers. More information will be provided next journal entry.

Copy number variation and disease This project looks at the discovery and assessment of the clinical significant of copy number variation detected in patients with a wide range of disorders, including mental retardation, epilepsy, autism, developmental and dysmorphism. My role in this project was to setup master mix and run gel for 2000 PCR products to identify the genotype wither heterozygote or homozygote.

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Heterozygous (+/-) means that segment of DNA having two different alleles. So that mean, each gene has two different copies; one is inherited from the father and the other from mother. When PCR products run on gel electrophoresis, bands were observed by using UV light. However, possessing two identical alleles means homozygous (+/+).

Genotyping identification assay is mainly based on PCR technology whether traditional or real time PCR. Murduch children research institute (MCRI) uses the traditional PCR that involves the using of UV light. This technique is not automated and requires post PCR processing (gel electrophoresis); therefore, it is time consuming. Moreover, the results are based on size discrimination. However, for each and every PCR application, optimal reaction conditions need to be ensured for the successful amplification of the PCR product. The basic reaction mixture consists of all the components necessary to make new segment of DNA in the PCR process template DNA, primers (forward and reverse), reaction buffer; magnesium chloride (MgCl2), dNTPs and aheat stable DNA polymerase.

A 25l PCR reaction mix was set up as provided below

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PCR1_Mastermix and PCR conditions

Final conc. 1x(1.5mM MgCl2) 1mM(2.5mM) 0.20mM 0.2uM 1U

Reagents dH2O 10xBuffer (15mm MgCl2) MgCl2 (25mM) dNTPs (10mM) PCR1_ FP+RP (10uM) Hot Start Tag DNA

x1 19.30 2.50 1.00 0.50 0.50 0.20 24.00 1.00

PCR Conditions

95C/15min 95C/30sec 60C/30sec 72C/45sec 72C/5min 4C/10min

40 cycles

Total volume

25.00

PCR Troubleshooting and gel electrophoresis: The Template DNA The amount of DNA in PCR has a significant effect on the outcome. So, using high amount might lead to poor DNA synthesis or false priming. The appropriate concentration for this experiment is found to be 1uL. Inadequate dNTPs Wrong concentration of dNTPs can cause problems for the outcome. Insufficient dNTPs concentration will inhibit the formation of the PCR product. So, after several trials to optimize the concentration of dNTPs, the appropriate concentration for this experiment is found to be 0.2mM. Primer Concentration The recommended concentration of primer is 0.25 mM for this procedure. The use of high concentration of primer usually leads to: o Raising the primer concentration can lead to formation of primer-dimers. So, after analysis through gel electrophoresis revealed that the PCR worked well. However there was presence of possibly some primer-dimer formation. Primerdimers could be a result of too many amplification cycles. Also, it could be a

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result of

the annealing cycle length ,

annealing temperature and

the

concentration of Mg2+. Gel electrophoreses: Electrophoresis is a procedure in the lab that sorting of the DNA based on the size and the charge using electric field. However, we loaded the DNA samples in negative end and that because DNA samples are negatively charged. As the electric reactions occurring the negative current it is going to push the samples toward the positive end and we end up with different size of fragment distributed through the gel. The smallest fragments are going to move faster and travel farthest and those fragments that are large in size are not going to travel as far. I have encountered in some problems while gel preparation and DNA loading: No bands or faint bands on the gel as results of: I. II. III. insufficient amount of DNA loaded on the gel. The DNA was degraded and appearing as smears on the gel. Improper electrophoresis condition. Maintain the voltage and dont allow the voltage exceed 90 V/cm. DNA ran off gel, timing is very important to avoid losing samples and make sure that the gel is immersed completely in next run and run for appropriate amount of time. DNA diffusion in the gel; you need to avoid long term storage before taking picture. As this may cause DNA diffusion and give low band intensity. Gel shift, you need to make sure that the gel take a proper position in gel casting tray. Bubble formation in gel wells, getting rid of bubbles can be removed by pipette tip. Poor formation of the wells; to avoid this matter by removing the comb after the gel become solid or completely polymerization. Thereafter, pour the buffer onto the gel and finally gently remove the comb.

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Optimization the condition for the real time PCR: Introduction: The real time PCR is based on probe chemistry which is essentially the quantitation and detection of fluorescent reporter molecule. Real time PCR measures the kinetics of the reaction
at the early phases where the target is first detected as it amplifies in the beginning, offering high precision and sensitivity, with the ability to detect as little as three fold changes in a reaction as compared to an end point PCR where the resolution is about tenfold. A fluorescent reporter molecule is used to monitor the progress of the amplification reaction where the fluorescence is measured by Sequence Detection Systems instruments. There are different detection chemistries available for use in real time PCR and choosing the most appropriate one depends on the overall objective and requirements of the research project. The following are the most commonly used detection chemistries:

A)DNA binding dyes as SYBY green:

SYBR greens are DNA binding dye. When the SYBR is added to the sample, it will binds with only double strand DNA and emit fluorescence. The increase of the fluorescent is direct

proportional to the amount of PCR product after each complete cycle. The cycle, at which the signals exceed a detection threshold cycle (Ct), it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The parameter Ct is known as the cycle number which the fluorescent emission exceed the fixed threshold as shown in the diagram below.
Signal intensity

Plateau phase

Ct Exponential phase

threshold

Ct1

Ct2

Cycle no

B) Probe based chemistries such as linear probes (TaqMan probes)

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TaqMan assays are designed using robust algorithms for detection for QPCR. Also, the probe which used in this assay is fluorogenic probe to detect the accumulation of the PCR products.

Standard Curve:
For the real time you need to setup standard curve from a control sample. Basically, it is a series dilution of control of known concentration. It is used to measure the value of unknown concentration when the samples are compared to the standard curve. Standard curve was diluted as shown in diagram below.

7.5ul

7.5ul

7.5ul

7.5ul

7.5ul

7.5ul

7.5ul

+ 7.5ul of dis H2O

Interpretation of results: In the end of each reaction, I rely on software program using the recorded signal intensity and applied: Rn+ is the value of reaction containing all mixtures (the sample of interest); Rn- is the value measured in NTC (baseline value). DRn is the difference between Rn+ and Rn-. The DRn is plotted against cycle numbers that produces the amplification curves and gives the Ct value.

I have tested different gradients to end up with optimization of the real time PCR as provided below.

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Reagents dH2O Buffer A MgCl2 dNTPs Betaglobin FP Betaglobin RP SRY FP SRY RP Betaglobin Probe SRY Probe AmpliGold Taq DNA Total volume

x1 0.7 ul 1 ul 2.5ul 0.75ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.05ul 2ul 10ul

Common problems that I experienced in using real time PCR:

I encountered several problems during setting up the master mix for the real time PCR. For example, probe which has been used in this method is very sensitive to the light. In first mixtures that have prepared was exposed to light for long time. In this case, the light is harmful and lead to degraded probe. So we end up with unusual amplification in some well and no amplification at all in others.

To avoid this problem, you need to make sure that the probe reagent is fresh prepared and covered well to reduce the light sensitivity.

In addition, I have experienced a problem with gene quantitation such as abnormal amplification or having no curves at all. Abnormal amplification plot during this experiment was noted. For example, amplification occurred later than expected:- possible cause: o You might not have enough templates. To be under control this factor you need to increase the amount of the DNA template.

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Amplification of the No template control (NTC). So, if you got amplification in NTC that may have a problem which is a contamination of the reactions mixture by DNA.

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DNA extraction: DNA extraction is simply defined as removal of the deoxyribonucleic acid from cells in which it normally resides. In this lab, DNA extraction is often an early step in many diagnostic processes such as screening genetic disorders.

How does it work? There are two different techniques that mainly used in MCRI to extract the DNA as shown below: Automated DNA extraction using Magna pure extraction machine and the DNA is usually extracted from cell lysate which has advantage as high throughput and low quality as disadvantage.

Manual DNA extraction using the column method (Qiagen kit) and the DNA is usually extracted from blood which has advantage as high quality and low throughput and time consuming as disadvantages.

Procedure undertaken: Qiagen purification kit:Qiagen plasmid purification kits are based on selectivity of patented resin that allowing purification of ultrapure DNA with high yield. The protocol is based on a modified alkaline lysis procedure. Thereafter, followed by binding of DNA to Qiagen anion-exchange resin under appropriate low salt and pH conditions. Proteins, RNA, lipids and low molecule weight are removed by wash buffer.

Determination of yield: NanoDrop product has played a pioneering role to measure the concentration of DNA. Absorbance at 260nm provides a measure of the concentration of the DNA samples. Good quality of the DNA was determined, by reading the absorbance at 260/280nm which is 1.8 0.1 for pure quality of DNA solution and decrease in the value evidences contamination by proteins.

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Common problems that I experienced in using Qiagen purification kit:

The main issue that I have experienced during this experiment was poor yield and quality. This problem can be caused by different factors that including:

Alkaline lysis inefficient which occurred when having large amount of cultured medium. So, this may result in poor lysis condition and gave low yield or no DNA, because the volumes of lysis buffers are not sufficient. Another possible reason is that insufficient mixing of lysis or doubling the volumes of those lysis buffers will result in low yield.

Also, it might be incorrectly prepared of the lysis buffers. The preparation of lysis buffers should be prepared according into the guide line of the kit to increase DNA yield and quality.

Inappropriate salt or pH conditions in buffers. In this case you need to double check that any buffers prepared in the laboratory were prepared according to the instructions provided with kit.

Salt concentration is to high in the sample; you need to dilute the sample and redissolve it. Colum was over loaded with DNA, use a bigger column. Colum blocked; use large volume of buffer and use prolonged centrifugation. to get supernatant. Then mix the sample with equilibration buffer.

Detailed description of the procedures, importance, and technical aspects of DNA methylation methods will be provided in the entry report.

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DNA methylation: DNA methylation is a naturally occurred in both eukaryotic and prokaryotic cells. In prokaryotes DNA methylation has played a pioneering role to protect host DNA from digestion restriction enzymes that are designed to attack foreign DNA. However,

eukaryotes DNA methylation functions in the regulation and control gene expression. The aberrant DNA methylation is phenomenon in cancer and it has been shown to play a fundamental role in embryonic development, gene imprinting, cell cycle regulation and Xchromosome gene silencing DNA methylation consists of the addition of a methyl group to the fifth carbon position of cytosine base via methytransferase enzyme. Procedure undertaken: EZ DNA Methylation-Gold Kits: The ability to detect and quantify DNA methylation efficiently has become essential for the study of cancer, genetic disease and gene expression. Also, there are number of methods that have been used to detect and quantify DNA methylation including: methylation-sensitive arbitrarily primed PCR and high-performance capillary electrophoresis. However, EZ DNA Methylation-Gold Kits used the bisulfite conversion method. It is based on treating

methylated DNA with bisulfite, which convert un-methylated cytosines into uracil. Once converted, the methylation profile of the DNA can be determined by PCR and followed by DNA sequencing.

The kit was very simple to follow, and it is one of the reasons that scientist choosing this technique.

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Seminars I have attended tow different seminars during my work placement. These seminars are held weekly on every Tuesday from 12 to 1pm. A) Fragile X: Fragile X syndrome is the most common cause of inherited intellectual disability worldwide. It can be found in all races and the latest statistics indicate that approximately 1 in 4000 males and 1 in 8000 females are affected. Usually the symptoms include global development delay and speech and communication problems. The name of this syndrome comes from its location on the X chromosome. In those patient that are affected by fragile X, it is usually to see feature at the end of X chromosome or its appeared narrowing at the end of X chromosome by using high power full microscope as shown below.

fra(X) female

Fra(X),Y male

However, Fragile X is caused by mutation of the FMR1 gene on the X chromosome. There are different repeat range of fragile X as shown below: FMR1 gene contains between 5-46 repeats of the CGG codon normal range 47-59 repeats: Grey zone, this subgroup dont have fragile X. 60-200 repeats: seen in some men and women and its called permutationPM. PM, the FMR1 gene is still working even when it contained a medium repeat.

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>200 repeats: seen in men and women affected by fragile X syndrome and this long expansion is called full mutation. In this case men who have full mutation in their gene , will have fragile X syndrome.

The expansion of the CGG trinucleotide is a result in a methylation with the 5-untranslated region of gene. This methylation of the FMR1 locus is located in Xq27.3 which believed to result in construction of the X chromosome. Mutation on this region can lead gene encodes and resulting in no protein production of FMRP which is essential for normal neuronal

functioning. FMRP plays important roles in learning and development of memory; also it shows to be involved formation of synapses. Test for FMR1 It can be identified the change on X chromosome, using a microscope karyotyping. This method is not precise as it appears in some cases to have fragile X syndrome in FMR1 faulty gene. However, genetic testing has shown to be more reliable to identify the X syndrome by looking at the number of CGG code repeats and their methylation status using southern blot.

Currently there is no cure for fragile X syndrome. However, there are special therapies (behavior therapy) and all methods of teaching and medication that provide a benefit to people with fragile X.

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B) DNA microarray:

DNA microarray is a common method used in cytogenetics laboratories to detect chromosomal abnormalities such as genomic copy number variation and single nucleotide polymorphism. Copy number variation can be duplication or deletion and usually caused by chromosomal rearrangement process such as translocation and inversion. Copy number variation and single nucleotide polymorphism are known to be associated with some types of cancers and some development abnormalities in children. Traditional methods for detection of copy number variation include FISH and chromosomal karyotyping. However, recently DNA micro array has been used extensively because it provides higher resolution and cover completely of the genome.

At the Victorian clinical genetics services (VCGS) are Affymetrix array. The Affymetrix DNA chip array used at the VCGS contains 2.5 million probes that cover huge parts of the genome and give more information at high resolution about any CNVs or SNPs.

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Staff meetings: Staff meetings considered to be an essential procedure in the laboratory, its an effective way to share common matters and to bring staff members up to date about the important changes within the laboratory.

In these meetings the management discusses topics and different information from around the lab. And its usually started with general administration discussion Thus; the staff will be informed of all the new events and changes occurring inside the laboratory.

Taking a closer look to the cytogentics section of VCGS they have a staff meeting every other Friday with the entire crew, and for these meetings there is sure to be a special agenda. In addition the agenda is flexible and changes can be made in the moment, and any employee who would like to add a point to the meeting, it can be added to issues for cytogentics meeting white board and the point will be discussed by the one they were added by and then by the group to get more views.

For example of the positive change resulting from an issue raised in a staff meeting was concerning the quality of stationary equipments for a new building the issue was noted by a senior member of the staff and because it had persisted for a few weeks, so it wasnt just a single person or a set of slides, and because the group agreed it wasnt a persons performance reasons were looked at to solve this matter.

Thus, staff meetings can improve the quality of work and getting accurate results inside the laboratory.

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Assay development for superior detection of sex chromosome aneuploidies: The aim of this study was to evaluate the specificity and sensitivity of the real time PCR method for sex determination and chromosome anaiploidies. The human sex determining region on the Y chromosome has been identified and the gene has been named as SRY gene. The normal function of SRY gene is to provide instructions to make a particular protein which called the sex determining region Y protein. This protein binds to specific region of DNA and control the activity of gene.SRY protein causes a fetus to develop as male. Normal people have 46 chromosomes; two of the 46 chromosome are called sex chromosome, known as X and Y. sex chromosome determine whether a fetus will develop female or male characteristics. Female have XX and male have XY. Procedure undertakes: DNA samples were extracted from blood and saliva specimens subjected to real time PCR. Amplification of SRY as a Y chromosome marker and globin as an interval control (2copies). The DNA test involves the amplification of specific region of SRY gene. The present of SRY amplified product directly indicates the presence of Y chromosome. Also, if there is an extra copy from Y chromosome as in some sex-linked disorders, the amplification showing double intensity as compared to the normal male control. Otherwise, it shows 0 intensity as in female on turners sample (X) or present same amount of intensity in normal male sample. For the real time you need to setup standard curve as mention before to measure the value of unknown concentration. The PCR cycle at which fluorescence exceeds background and a significant increase in fluorescence is observed is the threshold cycle. The Ct value is correlated with the starting template amount since it corresponded to PCR product accumulation. The quantification of the initial copy numbers of the unknown samples are facilitated by comparing the Ct values of the unknown samples to the standard curve.

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Standard curve for both gens are obtained and provided as shown below:
Detector: SRY Detector: Betaglobin

These figures show the standard curve for SRY gene and -globin gene. A dilution series of a control template of known concentration is prepared. Standard curve is generated by plotting the log of the unknown concentration against the Ct generated for each of known concentration standard dilution.

The detection of sex chromosome in plasma or saliva by real time PCR method has potential application for the diagnosis of sex linked disorders. Common problems in Real time PCR that I have experienced:

A common problem that I experienced with quantiation experiments including: Abnormal amplification possible cause: o You might not have enough templates. To be under control this factor you need to increase the amount of the DNA template. such as amplification occurred later than expected:-

NTC Positive Amplification; you have amplification in NTC can be caused by a contamination of reaction by DNA or RNA. Another cause can be one or more

reagents are contaminated. To overcome those problems are to use clean working area and make sure that the surface area is wiped by ethanol before and after started or use different tips after each aliquot.

No Amplification: There is no product, something is wrong with the master mix. Possible reason; probe concentration too low or matrix addition of DNA template wrong.

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Poor PCR Efficiency: Slope of the standard cure indicates that the PCR efficiency. It can be calculated by using software programs. The slope of PCR should be close to 3.3 as possible to give high efficiency. High efficiency gives later Ct at high concentration. There are numbers of factors can affect the efficiency such as inaccurate reagents and sample pipetting, probe, primer might need to be optimal or samples might contain PCR inhibitors.

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BACs-on-Beads assay: Summary of the tasks performed and observations: I have observed the procedure and the setup of the BoBs kit for 100 samples to detect the gains or losses of DNA in chromosomal regions. Overview: The new development assay for detection of losses and gains of chromosomal region is covered by KaryoLite BoBs kit. It is a quantitative test and belongs to BACs on-Beads family products. Moreover, this kit utilizing probes that derived from bacterial artificial chromosome. KaryoLite BoBs kit consists of 90 DNA probes which can be distinguished by Luminex instrument system. Principle: The detection of gain or loss in the regions of extracted DNA to which the clone mix can hybridize. After hybridization, the mean fluorescence intensity of the sample that hybridized to a specific probe is compared to the reference DNA tagged to a specific probe. BoBsoft analysis software is used to compare the intensity between samples and references(1).

Main steps of the BoBs kit as shown below:

DNA labeling Hybridization

DNA

Biotin labeled DNA

Purified biotin labeled DNA

DNA purification

DNA hybridizd to beads

Beads ready for analysis

Analysis

Results

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Briefly, sample and reference DNA are labeled by biotin. The labeled DNA is purified and the purified DNA with probes is hybridized for overnight. DNA washed and transferred onto filter and washed again and then incubation step with reporter. Thereafter, analysis steps using Luminex instrument to measure the amount of DNA bound to beads. Common problems in BoBs kit:
There are few problems with the kit. It is very robust. If you have very poor quality DNA or very small amount of DNA then the sample result will be noisy. It can detect to 50ng of DNA for the karyolite kit, but the company recommends starting with over 150ng preferably. The kit suggests you start with 240ng per patient sample of DNA.

Low yield of labeled DNA; you need to increase the amount of DNA starting. Make sure that you measure the amount of labeled DNA before the purification step. Cannot detect the labeled probes; DNA might be lost in purification step after labeling.

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Conclusion: The human sex determining region on the Y chromosome has been identified and the gene has been named as SRY gene. The normal function of SRY gene is to provide instructions to make a particular protein which called the sex determining region Y protein. This protein binds to specific region of DNA and control the activity of gene.SRY protein causes a fetus to develop as male. Genotyping identification assay is mainly based on PCR technology whether traditional or real time PCR. Murduch children research institute (MCRI) uses the traditional PCR that involves the using of UV light. This technique is not automated and requires post PCR processing (gel electrophoresis); therefore, it is time consuming. Moreover, the results are based on size discrimination. However, for each and every PCR application, optimal reaction conditions need to be ensured for the successful amplification of the PCR product. The basic reaction mixture consists of all the components necessary to make new segment of DNA in the PCR process template DNA, primers (forward and reverse), reaction buffer; magnesium chloride (MgCl2), dNTPs and aheat stable DNA polymerase.

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