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Summary Learning Objectives Molecular Diagnostics
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The learning objectives given in the course of molecular diagnostics.
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Test Bank for Molecular Diagnostics Fundamentals, Methods, and Clinical Applications, 3rd Edition, Lela Buckingham.
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Test Bank for Molecular Diagnostics Fundamentals, Methods, & Clinical Applications, 1st Edition, Maribeth L. Flaws, Lela Buckingham.
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Learning objectives Molecular DiagnosticsStudy task 1
Chapter 1: DNA
1. Describe the structure of DNA and nucleotides
Deoxyribonucleic acid (DNA) is a macromolecule of carbon, nitrogen, oxygen, phosphorous and
hydrogen atoms. It is assembled in units of nucleotides that are composed of a phosphorylated
ribose sugar and a nitrogen base. There are four nitrogen bases called nucleosides (bound to an
unphosphorylated sugar): adenine (A, purine), cytosine (C, pyrimidine), guanine (G, purine) and
thymine (T, pyrimidine), where C binds G (three hydrogen bonds) and A binds T (two hydrogen
bonds). These nitrogen bases are attached to a deoxyribose sugar, which forms a polymer with the
deoxyribose sugars of other nucleotides through a phosphodiester bond. Linear assembly of the
nucleotides makes up one strand of DNA, two strands comprise the DNA double helix. This helical
structure of DNA results from the physicochemical demands of the linear array of nucleotides in the
strand, as well as the surrounding chemical micro-environment, can affect the nature of the DNA
helix.
2. Describe the process of DNA replication and the function of the enzymes involved
The two DNA strands that make up a double helix have an antiparallel orientation. The DNA is
synthesized in the 5’ to 3’ direction by DNA polymerase, an enzyme (reads in the 3’ to 5’ direction)
that uses a template to determine which nucleotides to add to the chain.
Firstly, the double helix must be unwound by helicase, so that both strands can serve as a template.
The single strands are then elongated by hydrogen bonding of the complementary incoming
nucleotide to the nitrogen base. While
DNA replication on the leading strand
(3’ to 5’) proceeds in a continuous
manner, lagging strand (5’ to 3’) is
replicated in a discontinuous manner
with the use of Okazaki fragments
(small DNA fragments) by jumping
ahead a short distance (~1000 bases)
and then copies backwards.
DNA cannot be synthesized the novo; a preceding base must be present to provide the hydroxyl
group (deoxyribose 3’-hydroxyl oxygen). This base is provided by another enzyme called primase, a
RNA-synthesizing enzyme that catalyzes the synthesis of short (6 – 11 bp) RNA primers required for
priming DNA synthesis. Primase works repeatedly on the lagging strand to synthesize the Okazaki
fragments.
3. List the enzymes that modify DNA and state their specific function
• Polymerase, catalyzes the formation of phosphodiester bonds
• Helicase, unwinds and untangles DNA
• Primase, synthesizes a short RNA primer to prime DNA synthesis
• Methyltransferase, adds a methyl group to a nitrogen base
• Deaminase, takes an amino group from a nitrogen base
• Ligase, catalyzes the formation of a single phosphodiester bond
• Protease, degrades proteins
• Ribonuclease, degrades RNA
• Exonuclease, remove bases from the ends of DNA strands
• Endonuclease, cut DNA strands internally
- Restriction enzymes → blunt and sticky ends
, - Star activity → altered specificity, binding and cutting sequences other than the
expected recognition sequences.
4. Describe the process of conjugation and 5. Compare transformation/transfection,
transduction and conjugation
Conjugation
Transduction
, Transformation
Transfer of
DNA from one
organism to
another
without
protection of a
conjugative
bridge or viral
coat.
*Transfection
in animal cells
Chapter 2: RNA
6. Compare and contrast the structure of RNA with that of DNA
- DNA has deoxyribose sugars; RNA has ribose sugars
- DNA has thymine; RNA contains uracil
- DNA is double stranded; RNA is single stranded
7. List and compare the different types of RNA present in the cell
- mRNA
- rRNA
- tRNA
- small nuclear RNA (functions in splicing)
- hnRNA
- miRNA
- siRNA
- dsRNA
8. Describe the process of transcription (RNA replication) and the functions of the enzymes
involved and compare it to DNA replication (eukaryote)
mRNA carries the information in DNA to the ribosomes, where it is translated into proteins. RNA
transcription is catalyzed by RNA polymerase (interphase).
- Works more slowly than DNA polymerase
- Less fidelity
- Doesn’t require priming
- Polymerized in 5’ to 3’ direction, the first ribonucleoside triphosphate retains all
of its phosphate groups and subsequent ribonucleoside triphosphate retain only
alpha phosphate (closest to ribose sugar).
- Three types of RNA polymerases in eukaryotes (one in prokaryotes).
• RNA polymerase I → synthesize noncoding RNA in nucleolus
• RNA polymerase II → synthesizes mRNA in the nucleus
• RNA polymerase III → synthesize noncoding RNA in the nucleus
RNA polymerase starts at the promoter (initiation) using one of the DNA strands as a template, only
replacing the A-T bond for a A-U bond (elongation). This proceeds until a polyadenylation signal
(polyA site) is encountered. As the polymerase proceeds past the polyA site, the nascent mRNA is
released by an endonuclease associated with carboxy terminal end of pol II.
- RNA synthesis after this part is bound by exonuclease and degraded
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