Subject: PCR Techiques

Hi all! =) This is Lyn here. I will be the first in my group to share with you guys (and my group mates) about the things that i have done this first week of my attachment.

I'm attached to the research lab doing research relating to pharmacogenetics. My MP will be on that as well. Pharmacogenetics is basically the study of how the variability of the genetics of individuals affect the reponse to drug dosage. This is because, if an individual has a variant in his/ her gene, he/ she might need a lowerd drug dose as compared to an healthy individual with no variants. Thus, pharmacogenetics enables the prescription of the correct drug dose to the patient so as not to result in drug toxicity.

Bascially, what I do in the lab for this one week was preparing the PCR products using different primers, cast 1.5% and 2% agarose gel to carry out gel electrophoresis and the PCR which includes primer optimisation. Doing primer optimisation is actually to find out the optimum temperature whereby primers anneal to the DNA sample and result in bright sharp thick bands. I also get to do gel check for the PCR products that are prepared. It is similar to what we were shown during Mgen or Mbio practical sessions. However, in the lab, they use this machine known as the Gel-Doc to visualise the bands. In this machine, you place your completed agarose gel from electrophoresis process and turn on the UV so as to 'bring out' the bands. This machine is connected to a computer whereby there's a programme where you can print the video print of your agarose gel out. It comes out as a photo.

I also learnt about purification and sequencing. Purification is done actually to remove the excess primers and dNTPs that would interfere with the sequencing procedures. In purification, Exo-SAP is added to the PCR products. Exo = exonuclease 1: it cleaves and remove the excess primers. SAP = shrimp alkaline phosphatase: it cleaves and dephosphorylates the dNTPs so that they are unable to bind and carry out elongation.

Sequencing is done to determine the actual sequence of the DNA that is prodcued during the PCR that is complementary to the template DNA strand. This is done by adding the primers and the dye that contains dNTPs, ddNTPs and Taq polymerase. These ddNTPs are fluorescently labelled so that they can be detected and the sequence of the DNA strand complementary to the temlate DNA strand can be known. After the addition of the reagents needed, the samples are sent to the analyser, whereby the analyser will analyse the different ddNTPs that binds and also produce the peaks.

These are bascially what I have been doing for the first week of attachement.

Have fun at work for SIP. Take care everyone. =)

-Lyn-

What is CAP accreditation and CAP Lab Accreditation Programme?

CAP is an authorized accrediting organization1 that promotes excellent pathology and laboratory medicine practices to ensure welfare of its customers are taken care of.1, 2

Upon completing and passed the inspection programme done by CAP, a laboratory will be granted CAP accreditation. The laboratory is now proven to produce the best results.3

Laboratories can undergo CAP Lab Accreditation Programme to acquire CAP accreditation. The programme is authorised by CMS and recognised by JCAHO. It satisfies the requirements of different laboratory settings, comprises of a variety of disciplines and testing procedures, and enables high level of service to be obtained.3

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CMS: Centers for Medicare and Medicaid Services

JCAHO: Joint Commission on Accreditation of Healthcare OrganizationsReferences

1. METROPOLIS Health Services. (2005). College of American Pathologists [CAP]. Retrieved June 13, 2008, from http://www.metropolisindia.com/cap.asp

2. DNA Junction. College of American Pathologists (CAP) Laboratory Accreditation Program. Retrieved June 13, 2008, from http://www.dnajunction.com/accreditation/dna-cap.php

3. College of American Pathologists. (2008). About the Laboratory Accreditation Program. Retrieved June 13, 2008 from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=/portlets/contentViewer/show&_windowLabel=cntvwrPtlt&cntvwrPtlt{actionForm.contentReference}=laboratory_accreditation/aboutlap.html&_state=maximized&_pageLabel=cntvwr

Done by Lyn

Why is there a need for CAP accreditation?

By having a CAP accreditation, it can show that the laboratory has fulfilled the regulatory requirements, skilled in carrying out its specific tasks and that patient care and safety are always well taken care of to the highest possible quality, minimizing errors in laboratory results which can lead to wrong diagnosis and treatment.1,2 This thus, allows the existence of trust between the accredited laboratories and the third parties in the information generated during the testing procedures, preventing unnecessary verification that could save time and money.2 Furthermore, acknowledgement of test results generated in other countries can also be facilitated.2

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1. CLSI eNews. (2007). Excellence in Laboratory Performance: CLSI and CAP Aligned for a Common Goal. Retrieved June 14, 2008, from http://enews.nccls.org/clsi/textonly/2007-11-01/4.html

2. Singapore Accreditation Council. (2005). SAC E-ALERT-SAC-CAP Joint Programme. Retrieved June 14, 2008, from http://www.sac-accreditation.gov.sg/news.asp?month=7&year=2005

Done by Xin Yi

Benefits of CAP accreditation

CAP Laboratory Accreditation Program helps laboratories to meet and even exceed the requirements of the Centers for Medicare and Medicaid Services (CMS) and other Federal requirements as only CAP uses multi-disciplinary teams of qualified professionals to be inspectors. Under this program, CAP is able to attend the widest patient population and ensuring accurate and quality test results of patients. The laboratory standards are maintain by laboratories professionals, and from the self-inspection and participating in the inspection of other laboratories, the laboratory can learn the latest laboratory process and techniques.1,2

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1. College of American Pathologists. (2005). Benefits of CAP Accreditation. Retrieved June 13, 2008 from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_accreditation%2Flap_info%2Fbenefits.html&_state=maximized&_pageLabel=cntvwr

2. METROPOLIS Health Services. (2005). College of American Pathologists [CAP]. Retrieved June 13, 2008, from http://www.metropolisindia.com/cap.asp

Done by Ting Jie

Features of CAP System Inspection

A CAP System Inspection1 comprises of:

-utilization of standardized checklist and inspection dates for all sites

-integration criteria2 to ascertain that a common system is used to carry out inspections

-a pre-inspection meeting with a CAP inspection specialist to obtain relevant details and knowledge, and to assist the team leader in forming a team that is suitable in size and goal

-a CAP inspection specialist being present at the respective venues during the inspection to allow for a systematic management and communication

-allocation of one inspector to one inspection team of similar disciplines

-a post-inspection international consolidation meeting and a final report to determine system-level feedback

-a CAP system inspection document to indicate the laboratory’s accomplished performance

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1. College of American Pathologists. (2008). Features of a CAP System Inspection. Retrieved June 14, 2008, from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=/portlets/contentViewer/show&_windowLabel=cntvwrPtlt&cntvwrPtlt{actionForm.contentReference}=laboratory_accreditation/system_inspections_features.html&_state=maximized&_pageLabel=cntvwr
2. College of American Pathologists. (2006). Qualifying for a System Inspection. Retrieved June 14, 2008, from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_accreditation%2Fsystem_inspections_qualify.html&_state=maximized&_pageLabel=cntvwr

Done by Alexander

How is CAP Accreditation awarded?

CAP Accreditation is awarded when a laboratory has proven that it meets the criteria and standards for laboratory accreditation.1 A qualified inspector will inspect every 2 years, and both inspector and laboratory will evaluate its own standards by comparing against the standard CAP checklist.1 The assessment of areas includes laboratory equipment, computer services and the handling of specimens.2 Laboratories have to correct and improve on any errors detected, as unsatisfactory results will cause the laboratory to lose its CAP accreditation.1 Thus, CAP ensures that laboratories are of high standards, safe and have excellent procedures to produce sustainable and up-to standard results.2

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1. Hon Fon L. Mark. Setting the Standards for Cytogentics Laboratories. Medical Cytology. (2000). Retrieved June 15, 2008, from http://books.google.com.sg/books?id=VVn_Othsf0UC&pg=PA615&lpg=PA615&dq=how+is+CAP+accreditation+awarded&source=web&ots=cbXJDyjAaQ&sig=s6TJyLZ9klvl0ihqqS_aPGw1Kqo&hl=en&sa=X&oi=book_result&resnum=1&ct=result

2. DNA Junction. College of American Pathologists (CAP) Laboratory Accreditation Program. Retrieved June 15, 2008, from http://www.dnajunction.com/accreditation/dna-cap.php

Done by Lloyd