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Samenvattingen Moleculaire Diagnostiek - Jaar 3 - Minor Bioresearch
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Uitgebreide Moleculaire Diagnostiek samenvatting. Gebruik gemaakt van de lessen Moleculaire Diagnostiek en het boek.
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Samenvatting Moleculaire DiagnostiekTentamen in het Engels, MC en open vragen.
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Study task 1. Chapter 1: DNA, Chapter 2: RNA, Chapter 3: Proteins and Chapter 4: Nucleic Acid Extraction Methods. ........... 4
Chapter 1: DNA ................................................................................................................................................................... 4
Describe the structure of DNA and nucleotides...................................................................................................................... 4
Describe the process of DNA replication and the functions of the enzymes involved and List the enzymes that modify
DNA and state their specific function (e.g. polymerases, helicases, primase, methylases, deaminases, ligases,
exonucleases, endonucleases incl. star activity) ..................................................................................................................... 5
Describe the process of conjugation ....................................................................................................................................... 7
Compare transformation/transfection, transduction, and conjugation ................................................................................. 8
Chapter 2: RNA ................................................................................................................................................................... 8
Compare and contrast the structure of RNA with that of DNA .............................................................................................. 8
List and compare the different types of RNA present in the cell ............................................................................................ 9
Describe the process of transcription (RNA replication) and the functions of the enzymes involved and compare to DNA
replication ................................................................................................................................................................................ 9
Describe the cellular processing of messenger RNA ............................................................................................................. 12
Define cis and trans elements in relation to transcription ................................................................................................... 12
Describe gene regulation using the Lac operon as an example ............................................................................................ 12
Define epigenetics and describe examples of epigenetic phenomena ................................................................................ 13
Chapter 3: Proteins ........................................................................................................................................................... 14
Define primary, secondary, tertiary and quaternary structure of protein organization ...................................................... 14
Describe the process of translation and the functions of the enzymes involved ................................................................. 15
Describe how the genetic code must be read ....................................................................................................................... 16
Chapter 4: ......................................................................................................................................................................... 16
Distinguish between the isolation of total RNA with that of messenger RNA ...................................................................... 16
Describe the gel-based, spectrophotometric and fluorometric methods used to determine the quantity and quality of
DNA and RNA concentrations................................................................................................................................................ 18
Study task 2. Chapter 5: Resolution and Detection of Nucleic Acids, Chapter 12: Detection and Identification of
Microorganisms, Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins and Chapter 14: Molecular
Oncology. .................................................................................................................................................................................... 20
Chapter 5: Resolution and detection of Nucleic acids ..................................................................................................... 20
Explain the principle and performance of electrophoresis ................................................................................................... 20
Compare the agarose and polyacrylamide gel polymers commonly used to resolve nucleic acids and state the utility of
each polymer ......................................................................................................................................................................... 20
Explain the principle and performance of capillary gel electrophoresis as it is applied to nucleic acid separation ............ 20
Give an overview of buffers and buffer additives used in electrophoretic separation, including the constituents, purpose
and importance ..................................................................................................................................................................... 20
Describe and compare pulse field gel electrophoresis (PFGE) and regular electrophoresis techniques with regards to
method and applications ....................................................................................................................................................... 21
Chapter 12: Detection and identification of micro-organisms......................................................................................... 21
Describe and explain methods for molecular strain typing and interpret results (e.g. PFGE, RFLP, Southern blot) ........... 21
Chapter 6: ......................................................................................................................................................................... 22
1
, Construct a restriction enzyme map of a DNA plasmid or fragment .................................................................................... 22
Describe and diagram the Southern blot procedure northern blot and western blot procedure ....................................... 22
Explain depurination and denaturation of dissolved DNA .................................................................................................... 22
Discuss the purpose and structure of probes that are used for blotting procedures .......................................................... 23
Define hybridization, stringency and melting temperature and indicate how conditions affect stringency ....................... 23
Calculate the melting temperature of a given sequence of ds DNA ..................................................................................... 24
Compare radioactive and nonradioactive DNA detection methods ..................................................................................... 24
Compare dot and slot blotting methods ............................................................................................................................... 24
Describe microarray technology, its applications and compare it to classical blotting ........................................................ 24
Describe and explain Comparative Genomic Hybridization (CGH) ....................................................................................... 25
Chapter 14: Molecular Oncology ...................................................................................................................................... 25
Explain what microsatellite instability (MSI) is and how it can be detected ........................................................................ 25
Interpret microsatellite instability and loss of heterozygosity results .................................................................................. 26
Study task 3. Chapter 7: Nucleic Acid Amplification, Chapter 11: DNA polymorphisms and Human Identification, Chapter 12:
Detection and Identification of Microorganisms and Chapter 14: Molecular Oncology. .......................................................... 26
Chapter 7: Nucleic acid amplification ............................................................................................................................... 26
Describe and compare the following assays for amplifying nucleic acids: polymerase chain reaction (PCR), multiplex
ligation-dependent amplification (MLPA and transcription-mediated amplification (TMA) in terms of type of target
nucleic acid, principle, major elements of the procedure, type of amplicon produced, major enzyme(s) employed and
applications............................................................................................................................................................................ 26
Describe examples of modifications that have been developed for PCR (e.g. real-time PCR, ss-PCR, nested-PCR, multiplex
PCR, methylation specific-PCR) ............................................................................................................................................. 27
Differentiate between target amplification and signal amplification ................................................................................... 28
Chapter 11: DNA polymorphism and Human Identification ............................................................................................ 28
Describe short tandem repeat structure and nomenclature ................................................................................................ 28
Describe gender identification using the amelogenin locus ................................................................................................. 29
Interpret STR analysis results for different applications (e.g. parentage and engrafment testing) ..................................... 29
Chapter 12: Detection and Identification of micro-organisms ........................................................................................ 30
Describe and explain methods for molecular strain typing and interpret results(RAPD, AFLP etc.) .................................... 30
Chapter 14: Molecular Oncology ...................................................................................................................................... 31
Describe and explain gene rearrangements ......................................................................................................................... 31
Show how clonality is detected using antibody and T-cell receptor gene rearrangements................................................. 31
Study task 4. Chapter 8: Chromosomal Structure and Chromosomal Mutations and Chapter 14: Molecular Oncology ......... 32
Chapter 8: Chromosomal structure and chromosomal mutations .................................................................................. 32
Define mutations and polymorphisms .................................................................................................................................. 32
Distinguish the three types of DNA mutations: genome, chromosomal and gene .............................................................. 32
Describe chromosomal compaction and the proteins involved in chromatin structure ...................................................... 32
Diagram a human chromosome and label the centromere, q arm, p arm and telomere .................................................... 33
Illustrate the different types of structural mutations that occur in chromosomes .............................................................. 33
Describe how chromosomes are visualized using different staining techniques and name the resulting banding pattern 34
Describe and compare interphase and metaphase FISH analyses........................................................................................ 34
Describe the application of the different FISH probes .......................................................................................................... 34
Chapter 14: Molecular Oncology ...................................................................................................................................... 35
2
, Define oncogenes and tumor suppressor genes ................................................................................................................... 35
Identify checkpoints in the cell division cycle that are critical for regulated cell proliferation ............................................ 35
List molecular targets used for diagnosing and monitoring solid tumors and describe the techniques that are used (e.g.
the EGFR gene family) ........................................................................................................................................................... 35
Chapter 12: Detection and Identification of micro-organisms ........................................................................................ 36
Define classical and molecular methods of virus detection.................................................................................................. 36
Explain the differences between genotypic and phenotypic drug sensitivity testing .......................................................... 36
Study task 5. Chapter 11: DNA polymorphisms and Human Identification, Chapter 12: Detection and Identification of
Microorganisms and Chapter 13: Molecular Detection of Inherited Diseases .......................................................................... 37
Chapter 11: ....................................................................................................................................................................... 37
Compare and contrast different types of polymorphisms (e.g. VNTRs, STRs, SNPs, RFLPs)................................................. 37
Define restriction fragment length polymorphisms (RFLPs) and discuss how they are used in genetic mapping, parentage
testing and human identification .......................................................................................................................................... 38
Chapter 13: Molecular detection of inherited diseases ................................................................................................... 38
Define the terms polyploidy, mosaicism and chimerism ...................................................................................................... 38
Describe mendelian patterns of inheritance as exhibited by family pedigrees .................................................................... 39
Describe which techniques can be used to detect mutations in single-gene disorders ....................................................... 39
Define variable expressivity, genetic heterogeneity, and (incomplete) penetrance; and explain how these affect
inheritance patterns .............................................................................................................................................................. 40
Discuss non-Mendelian inheritance, and give examples of these types of inheritance, such as mitochondrial disorders
and trinucleotide repeat expansion diseases ........................................................................................................................ 40
Explain how genomic imprinting (epigenetics) can affect disease phenotype ..................................................................... 41
Study task 6. Chapter 9: Gene Mutations .................................................................................................................................. 42
Chapter 9: Gene Mutations .............................................................................................................................................. 42
Compare phenotypic consequences of different types of point mutations ......................................................................... 42
Discuss methods used to detect point mutations : hybridization based, sequence (polymerization) based, and cleavage
based (e.g. SSCP, DGGE, TGGE, melt curve analysis, heteroduplex assays, MLPA etc.) ....................................................... 43
Study task 7. Chapter 10: DNA sequencing ................................................................................................................................ 48
Chapter 10: DNA sequencing ............................................................................................................................................ 48
Describe and compare the chemical (Maxam/Gilbert) and the chain termination (Sanger) sequencing methods ............ 48
List the components and the molecular reactions that occur in Sanger chain termination sequencing and interpret results
............................................................................................................................................................................................... 48
Discuss the differences between dye primer and dye terminator sequencing .................................................................... 48
Derive a text DNA sequence from raw sequencing data ...................................................................................................... 49
Explain examples of alternative sequencing methods, such as bisulfite sequencing and pyrosequencing ......................... 49
Give 3 examples of Next Generation Sequencing (NGS) techniques and explain the advantages/disadvantages of NGS .. 53
3
,Study task 1. Chapter 1: DNA, Chapter 2: RNA, Chapter 3: Proteins and
Chapter 4: Nucleic Acid Extraction Methods.
Chapter 1: DNA
Describe the structure of DNA and nucleotides
DNA (deoxyribonucleïnezuur).
Bevat alle erfelijke informatie van een organisme
• DNA strengen zijn complementair
• DNA strengen vormen een dubbele helix
• De twee DNA strengen worden bij elkaar gehouden door
waterstofbruggen
Nucleotides: Base+Suiker+Fosfaat
Nucleotiden vormen een groep van bio-organische
verbindingen die de bouwstenen voor DNA en RNA
vormen.
Structuren:
Een nucleotide bestaat uit drie hoofdcomponenten:
een purine- of een pyrimidinebase (een
zogenaamde nucleobase)
Adenine, Cytosine, Guanine en Thymine
een suiker met vijf koolstofatomen (een
zogenaamd pentose)
één tot drie fosfaatgroepen
Nucleosides: Base+Suiker
Een nucleoside is een bio-organische verbinding die bestaat uit een suiker (pentose) met 5
koolstofatomen en een nucleobase (een purine- of een pyrimidinederivaat).
Ribose sugar: Ribose is een monosacharide.
Nitrogen base: Rol kan worden beschouwd als een bouwsteen voor DNA en RNA.
DNA: adenine (A), guanine (G) = Purine, thymine (T), and cytosine (C) = Pyrimidine
RNA: adenine (A), guanine (G), cytosine (C), and uracil (U).
Purine (2 ringen): Adenine en Guanine Pyrimidine: (1 ring): Cytosine en Thymine
4
,Samenvatting Moleculaire Diagnostiek – Minor Bioresearch 2017 – Joyce Bakker
De suiker en fosfaatgroepen zijn covalent verbonden en vormen de
ruggegraat van de keten
• 3’ einde: hydroxyl-groep van suiker
• 5’ einde: fosfaat groep
Hydrogen bonds (2 or 3): Een
waterstofbinding is een zwakke chemische
binding die optreedt tussen
waterstofatomen en meer elektronegatieve
atomen. (A)denin en (T)hymine = 2
waterstofbindingen, (G)uanine en
(C)ytosine = 3 waterstofbindgen
Double helix: De DNA dubbele helix
polymeer van nucleïnezuren,
bijeengehouden door nucleotiden die
basepaar elkaar.
Describe the process of DNA replication and the functions of the enzymes involved and List the
enzymes that modify DNA and state their specific function (e.g. polymerases, helicases,
primase, methylases, deaminases, ligases, exonucleases, endonucleases incl. star activity)
DNA replicatie: productie van
twee complete dubbele helixen
uit een helix.
DNA-replicatie is nodig voor de
celdeling (mitose). De replicatie
begint op vaste plaatsen op het
DNA, de zogenaamde 'origin of
replication'.
DNA strengen zijn complementair. Beide
strengen kunnen dienen als template.
Ondanks de antiparallel aar d van de twee
strengen DNA synthese verloopt langs beide
strengen in dezelfde richting (5’ naar 3’).
DNA replicatie is semiconservatief. Een
combinatie van strengen.
5
, Samenvatting Moleculaire Diagnostiek – Minor Bioresearch 2017 – Joyce Bakker
Replicatie origin: Bij de replicatie
origin verbreken initiator eiwitten de
waterstofbruggen tussen de DNA
strengen.
- Bacterieel genoom (circulair
DNA) heeft 1 replicatie origin.
- Menselijk genoom heeft ca.
10.000 replicatie origins →
verkort replicatie tijd
Leading strand: continu gevormde DNA streng. 5’ 3’
Lagging strand: discontinu gevormde DNA streng. 3’ 5’
Okazaki-fragment: DNA streng die groeit aan de 5’ kant wordt discontinu gemaakt in kleine stukjes (Okazaki
fragmenten). Okazaki fragmenten worden later verbonden.
RNA-primer : stukje RNA geproduceerd door primase (RNA polymerase) dat dient als begin voor DNA synthese
Primase: Maakt RNA-primer. Primase kan twee losse nucleotiden verbinden.
1. Enzym helicase ontwind het DNA helix. Breekt het waterstof binding van het DNA backbone.
2. Enkelstrengs bindings eiwitten(SS=Single Stranded) zorgt dat terug binding niet plaats vindt.
3. Het leading strand template die van 5’ naar 3’ loopt wordt gemaakt m.b.v DNA polymerase III
4. De lagging strand template die van 3’ naar 5’ loopt wordt in segmenten gevormt, namelijk Okazaki-
fragmenten.
5. RNA primase legt een RNA primer neer.
6. DNA polymerase III legt de gecomplimenteerde baseparen neer. (P III vervangt het RNA primer)
7. DNA Polymerase I verwijderd de Okazaki fragmenten
8. DNA ligase bindt de Okazaki-fragmenten met elkaar.
Replicatie machine: Replicatie van DNA wordt uitgevoerd door de replicatie machine (eiwit cluster), DNA
polymerase (maakt nieuwe DNA keten)
Helicase: gebruikt energie uit ATP hydrolyse om de dubbele helix te openen
Primase/ RNA polymerase: Maakt RNA-primer.
Sliding clamp: Houdt DNA polymerase gebonden aan de DNA streng
Clamp loader: Plaatsen van de sliding clamp door hydrolyse van ATP. Eenmalig op de leading streng. Voor elk
nieuw Okazaki fragment op de lagging streng.
Enkelstrengs bindings eiwitten: binding aan enkelstrengs DNA om vorming van dubbele helix te voorkomen.
DNA polymerase 1: Deze reparatie polymerase is betrokken bij excisie met 3'-5 'en 5'-3' exonuclease activiteit
en verwerking van Okazaki-fragmenten die tijdens achterblijvende streng synthese.
DNA polymerase 2: De belangrijkste rol van Pol II wordt dat het vermogen om polymerase-activiteit te richten
op de replicatievork worden geïnstalleerd en hielp Pol III bypass terminal mismatches.
DNA polymerase 3: is het primaire enzym betrokken bij DNA-replicatie.
Polymerase: is een enzymcomplex betrokken bij de DNA-replicatie.
Helicases: ontspannen en te ontwarren
Primase: synthetiseert een korte ribonucleïnezuur (RNA) naar primer DNA-synthese
Methylasen: voeg methylgroepen aan stikstofbasen
Deaminases: neem aminogroepen van stikstofbasen
Ligases: katalyseren de vorming van een enkele fosfodiesterbinding
Nucleases zijn Exonucleasen: verwijder bases van de uiteinden van de DNA-strengen en endonucleases:
knippen DNA strengen intern.
6
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