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Genome Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Genome and links to relevant pages within the course.

Learn more about laboratory continuing education for medical technologists to earn CE credit for AMT, ASCP, NCA, and state license renewal and recertification. Or get information about laboratory safety and compliance courses that deliver cost-effective OSHA safety training and continuing education to your laboratory's employees.



Advances in Noninvasive Prenatal Testing For Down Syndrome and other Trisomies
Massively Parallel Signature Sequencing (MPSS): Cloning of cDNA Fragments on Microbeads

The MPSS technique occurs in two major steps. In the first step, in vitro cloning of cDNA fragments on microbeads occurs. cDNA fragments are cloned onto microbeads using the Lynx Megaclone technology. Starting with one million mRNA molecules from a particular cell or tissue sample, Megaclone will produce one million beads, each containing 100,000 cloned copies of cDNA from each mRNA molecule. All molecules are covalently attached to the microbeads at their poly(A) ends, so the DpnII end is available for the sequencing reactions. The image on the right illustrates the in vitro cloning of cDNA fragments on microbeads. The procedure involves both microbead tagged vector library preparation and sample preparation.Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine, http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml.

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Massively Parallel Signature Sequencing (MPSS): Sequencing Process and Analysis of Sequence Patterns

In step two, the microbeads are loaded and the sequencing process is initiated. Typically, five rounds produce about 16-20 signature sequence products. Several rounds of sequence determinations are performed and a sequence pattern or signature is identified from each microbead. The process is performed in parallel with about 1 million sequence signatures produced per overall assay. Each signature sequence is then analyzed, and compared with all other signatures and all identical signatures are counted. The level of expression of any single gene is then calculated.The image on the right illustrates the sequencing process and analysis of sequence patterns. Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine, http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml.The MPSS technique is quite complex. If you would like a more in-depth explanation, please click on the "More Info" button below and access the resources that are provided.

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References

Abnormalities of chromosome number (Aneuploidy). Available at: http://www.utmb.edu/pedi_ed/CORE/MedicalGenetics/page_11.htm. Accessed May 1, 2013.Aetna Clinical Coverage Policy, Serum Marker Screening for Down Syndrome, Policy #0464, reviewed 2/4/12. Available at: http://www.aetna.com/cpb/medical/data/400_499/0464.html. Accessed May 1, 2013.AFP test information. American Pregnancy Organization. Available at: http://americanpregnancy.org/prenataltesting/afp.html. Accessed May 1, 2013.American College of Obstetricians and Gynecologists. ACOG Committee Opinion No. 545, December 2012. Available at: http://www.acog.org/Resources_And_Publications/Committee_Opinions/Committee_on_Genetics/Noninvasive_Prenatal_Testing_for_Fetal_Aneuploidy. Accessed May 1, 2013. Aneuploidy: NCBI, US Library of Medicine. Available at: http://www.ncbi.nlm.nih.gov/books/NBK21870/. Accessed May 1, 2013.CAP Commission on Laboratory Accreditation- Laboratory Accreditation Program. Chemistry and Toxicology Checklist. Northfield, IL: College of American Pathologists: September 2012. Cigna Coverage Policy. Down syndrome screening. Policy #0211, reviewed 7/15/12. Available at: http://www.cigna.com/assets/docs/health-care-professionals/coverage_positions/mm_0211_coveragepositioncriteria_down_syndrome_screening.pdf. Accessed May 1, 2013. Definition of MSAFP (maternal serum alpha-fetoprotein. Available at: http://www.medterms.com/script/main/art.asp?articlekey=4446. Accessed May 1, 2013. Down syndrome: Genetics home reference. US National Library of Medicine, NIH. Pub. 1/17/13. Available at: http://ghr.nlm.nih.gov/condition/down-syndrome. Accessed May 1, 2013.Down syndrome genetic testing basics, Aetna Intelihealth, updated 6/28/11. Available at: http://www.intelihealth.com/IH/ihtIH/WSIHW000/32193/35641.html. Accessed May 1, 2013.Down syndrome. Mayo Clinic web site. Available at: http://www.mayoclinic.com/health/down-syndrome/DS00182. Accessed May 1, 2013. Down Syndrome: The ARC, 2013 update. Available at: http://www.thearc.org/page.aspx?pid=2546. Accessed May 1, 2013. Down Syndrome: The March of Dimes web site Information. Available at:http://www.marchofdimes.com/baby/birthdefects_downsyndrome.html?gclid=CIyNoJCP-rQCFQHonAodSnEAXg. Accessed May 1, 2013.Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing. California Technology Assessment Forum. [Draft]. Available athttp://www.ctaf.org/sites/default/files/u39/121017_draft_prenatal.pdf. Accessed May 1, 2013.MPSS technical information. National Center for Biotechnology Information, NCBI. Available at: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml. Accessed May 1, 2013. Morris JK, Mutton DE, Alberman E. Revised estimates of the maternal age specific live birth prevalence of Down's syndrome. J Med Screen.2002;9(1):2–6.Newberger DS. Down syndrome: Prenatal risk assessment and diagnosis. Available at: http://www.aafp.org/afp/2000/0815/p825.html. Accessed May 1, 2013.Resta RG. Changing demographics of advanced maternal age (AMA) and the impact on the predicted incidence of Down syndrome in the United States: Implications for prenatal screening and genetic counseling. Am J Med Genet A. Feb 15 2005;133A(1):31–36. Rodeck CH, Whittle MJ. Fetal Medicine: Basic Science and Clinical Practice. Philadelphia: Churchill Livingstone; 2009.Sietske NH, Perlstein D. Downs syndrome. Available at: http://www.medicinenet.com/down_syndrome/article.htm. Accessed May 1, 2013.Test ID: MAFP. Mayo Clinic Website. Available at: http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/81169. Accessed May 1, 2013.The Harmony Prenatal Test. Test information. Aiosa Diagnostics. Available at: http://www.ariosadx.com/about-the-science/. Accessed May 1, 2013. Torres TT, Metta M, Ottenwälder B, Schlötterer C. Gene expression profiling by massively parallel sequencing. Genome Res. 2007 Nov 21 PMID: 18032722. Triple screen testing. AACC Lab tests online. Available at: http://labtestsonline.org/understanding/analytes/triple-screen/tab/test. Accessed May 1, 2013.Tufts Health Plan provider policy. Genetic testing of maternal serum. Policy #2210630. Available at: http://www.tuftshealthplan.com/providers/pdf/mng/genetic_testing_maternit.pdf. Accessed May 1, 2013.Verifi Prenatal Test. Test information. Verinata Health. Available at: http://www.verinata.com/providers/provider-overview/. Accessed May 1, 2013.

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Fundamentals of Molecular Diagnostics (retired 2/12/2013)
Human Genome

Much research has been conducted to identify the alphabet of the human cellular language otherwise known as the human genome. This identification or roadmap of the human genetic material has opened the door to the mainstreaming of molecular diagnostics within the clinical laboratory setting.While the mapping of the human genome project is complete, many times it is not necessary to be able to identify the entire sequence; rather, we can use the specific portion of the code that is unique to the disease or condition in question. These short portions of the genetic molecular sequence or oligonucleotides, can then be used as probes to seek out and detect or amplify the target sequence.

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Terms and Definitions

TermDefinitionCodonA three nucleotide base sequence that codes for an amino acid Genome The genetic code composed of 64 codons that code for 21 amino acids and 3 stop codons (amino acids are the building blocks of proteins and stop codons stop the writing process much like a period at the end of a sentence)Nucleic acid Polymer made of monomers; two examples are RNA and DNATranscription Process of transferring information from DNA into an RNA messageTranslationThe formation of an amino acid from RNADeoxyribonucleic acid (DNA) A double-stranded polymer of nucleotides that houses genetic information Ribonucleic acid (RNA) Typically a single-stranded polymer that is much shorter than DNA but chemically similar with a few differences (eg - uracil replaces thymine)ReplicationReproduction of DNA content from parent to daughter cell during cell division Amplification methodsTechniques that increase the amount of the target, the detection signal, or the probe so that sequences are readily detectedFluorescenceThe emission of light at a longer wavelength when the light is excited at a shorter wavelengthOligonucleotideShort, single-stranded nucleic acidProbeA nucleic acid used to identify a hybridization targetPolymerase chain reaction (PCR)An amplification method performed in vitro

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The three base nucleotide sequence that provides the information necessary to identify an amino acid is termed a(n):View Page

HIV Safety for Florida
A retrovirus such as HIV produces ________ from _________.View Page
The protein component that surrounds the genome is called a:View Page
Function of HIV Genes

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene Number Abbreviation Gene Function 1 gag capsid proteins 2 pol viral enzymes 3 env envelope proteins 4 vif regulatory gene 5 tat regulatory gene 6 vpu regulatory gene 7 nef regulatory gene 8 vpr regulatory gene 9 rev regulatory gene

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Basic Structural Components

HIV consists of two basic components: a core of nucleic acid, called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The capsid is made up of subunits called capsomeres.

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HIV: Structure and Replication (retired 2/20/2013)
The protein component that surrounds the genome is called a:View Page
What is the source of the HIV envelope?View Page
The core (genome) of the HIV viron contains:View Page
Function of HIV Genes

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene NumberAbbreviationGene Function1gagcapsid proteins2polviral enzymes3envenvelope proteins4vifregulatory gene5tatregulatory gene6vpuregulatory gene7nefregulatory gene8vprregulatory gene9revregulatory gene

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Basic Structural Components

Human immunodeficiency virus (HIV) belongs to the Family Retroviridae and consists of two basic components: a core of ribonucleic acid (RNA), called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The HIV genome consists of three major genes: group specific antigens (Ags) or capsid proteins (gag); polymerase gene proteins: reverse transcriptase, protease, and integrase enzymes (pol); and envelope glycoproteins (env).The capsid is made up of subunits called capsomeres. Viral proteins are identified as either "gp" for glycoprotein or "p" for protein followed by the molecular weight in kilodaltons. For example, HIV-1 includes the envelope proteins gp160, gp120, and gp41; the gag core gene proteins, p55, p24, and p17; the polymerase gene proteins, p66, p51, and p31. HIV-2 proteins are similar to HIV-1 proteins. However, some of the proteins differ in molecular weight from those found in HIV-1.

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Penetration and Infection

After penetration of the cell membrane by the gp41, the HIV capsid enters the cell's cytoplasm. Next, cellular enzymes strip away the capsid so that the HIV genome is released. Also stripped away are proteins p24 and p17. Protein 24 coats the HIV genome and protein 17 lines the inside of the capsid.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
HPV Structure

Papillomaviruses are small DNA viruses that measure approximately 55 nm in diameter and belong to the family Papovaviridae. HPV is a non-enveloped virus and is composed of an icosahedral-shaped protein capsid enfolding a circular, double-stranded DNA genome of approximately 7,900 base pairs.Until recently, diagnostic laboratory testing for HPV was impossible since the virus does not grow in tissue cultures or in laboratory animals. Currently, with the recent technologic advancements in molecular biology techniques for HPV testing, scientists have isolated more than 100 different HPV types.

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HPV Viral Types

To differentiate one HPV viral type from another involves the detection of different genetic structures. In order to effectively type an HPV virus, there must be less than 90% DNA base-pairing homology in specific regions (usually coding areas) between the two types. In other words, one viral type has more than a 10% difference in its genetic structure compared to a genome of a different HPV virus type.Of the more than 100 HPV viral types that have been detected, approximately 100 have been fully identified and about 40 types are known to be associated with sexual transmission. Genital HPV types are divided into low-risk HPV and high-risk HPV categories.

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HPV Genome and Proteins

When HPV infects host cells, several HPV DNA-coded proteins initiate cellular changes. Two such areas in the genome are the open reading frames E1 to E7 and the late open reading frames L1 and L2. The proteins encoded by E1 to E7 regions of the genome are responsible for HPV-gene regulation and cell transformation. Proteins resulting from L1 and L2 form the viral shell.E6 and E7 encoded proteins are the most important HPV proteins in malignancy transformations. These viral proteins work together to convert normal host cells to malignant cells. E6 proteins interact with intracellular protein p53 and E7 proteins interact with intracellular retinoblastoma (Rb) protein. Intracellular proteins p53 and Rb regulate cellular growth. Both p53 and Rb are tumor suppressor proteins.When chromosomal damage occurs in normal cellular growth, p53 halts cellular growth and allows DNA repair enzymes to repair damage. Rb also halts cellular growth in DNA damage by inducing apoptosis (cellular death). When HPV E6 proteins bind to p53 and HPV E7 to Rb, mutations accumulate, unchecked cellular growth occurs, and a state of chromosomal instability results. This instability and unregulated cellular growth increases the chance of forming malignant cells. Viral E1, E2, and E5 encoded proteins may also damage cellular processes when HPV infects cells and can lead to malignant transformations.

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Select the statement that correctly describes the HPV genome and its endoded proteins.View Page
Carcinogenesis of Cervical Cancer Continued

Numerous genetic events occur over a relatively long period of time that lead to the development of cervical carcinoma. The protein products of tumor suppressor genes are the regulators of cell growth as discussed previously. Two intracellular protein products of tumor suppressor genes located within human cells are p53 and Rb. As noted earlier, protein products from HPV genes E6 and E7 bind to p53 and Rb, which results in unregulated cell growth. This unregulated growth prevents normal DNA repair, allowing for mutations to accumulate in the cell. As this process continues, it is postulated that a proto-oncogene becomes mutated, which in turn activates oncogenes.The E2 gene in HPV controls the production of E6 and E7 in the normal viral life cycle. When the viral genome is integrated into host cells, the E2 gene is disturbed and uncontrolled production of E6 and E7 protein products occurs. This leads to a greater interaction and disabling of host cell tumor suppressor gene products. The genes E6 and E7 of HPV Types 16 and 18 have a greater affinity for tumor suppressor gene products than other HPV types. This explains the greater virulence associated with Types 16 and 18 and their association with 70% of cervical cancer.

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Molecular Methods in Clinical Microbiology
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR.PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified. RT-PCR ProcessAfter RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.If the target is present, primers anneal to the RNA strand.Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.The temperature is raised to 95o C, and the RNA/DNA strands are denatured.The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.Polymerase enzyme synthesizes a new DNA strand, extending from the primer.Multiple cycles geometrically increase the number of copies of DNA.RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.

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Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics (retired 10/15/2012)
Pharmacogenomics Definition

Pharmacogenomics (PGx) is the study of how variations in the human genome affect a given individual's response to medications.

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Individualized Medicine

It has been said that we live in a new era of "individualized medicine." One of the primary drivers for this idea is the emerging field of pharmacogenomics (PGx). PGx is the study of how individual variations in the human genome affect responses to medications. The term "pharmacogenetics" is also used for this discipline (people in the field use both terms); however, the term 'pharmacogenomics' is becoming more popular since we now know the entire human genome. The primary reason that individuals metabolize and respond to drugs differently is the inter-individual differences in receptor proteins and enzymes that metabolize the drugs. Mutations in these receptor proteins and enzymes can give rise to very different responses to drugs. In PGx, these mutations are referred to as variants.

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Real-Time PCR
Reverse Transcriptase PCR (RT-PCR)

PCR can be modified for the amplification of RNA with one additional step prior to the PCR process -- the addition of a retrovirus enzyme called reverse transcriptase. Reverse transcriptase is used to create a copy of DNA using the original RNA specimen. Though there are thermostable polymerases that have reverse transcriptase capabilities, they are not commonly used. Reverse transcriptase PCR (RT-PCR) is used for the detection of viruses, such as HIV, that have an RNA genome. RT-PCR methods provide early detection of infection, even before the formation of antibodies. Therefore, it is a particularly useful method for HIV and hepatitis C virus(HCV) detection in blood bank nucleic acid testing. In addition to testing for HIV and HCV, RT-PCR is also used for detection of influenza A virus and other microorganisms where the target is RNA.RT-PCR is commonly combined with real time PCR.

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