7.
1 DNA Structure and Replication
DNA Structure
Outline how Franklin and Wilkins used X-ray crystallography to elucidate the structure of DNA
DNA was crystallised and then targeted with an X-ray (whose beam became diffracted by DNA crystals)
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The scattering pattern created by the diffracted X-ray was recorded on film
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This pattern was then analysed to elucidate the structure of DNA
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Describe, with the aid of the diagram, the organisation of DNA into chromatin within eukaryotic cells
DNA is wrapped around histone proteins to form nucleosomes
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Nucleosomes are grouped together (chromatosomes) and then arranged into fibres (chromatin)
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DNA is usually organised as chromatin within the nucleus, except during cell division (when the chromatin
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condenses to form chromosomes)
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Differentiate between euchromatin and heterochromatin
Euchromatin is more loosely packed and corresponds to active segments of DNA (i.e. active genes)
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Heterochromatin is more densely packaged and corresponds to inactive segments of DNA
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Different cells have different segments of DNA packaged as euchromatin and heterochromatin
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Outline the structure of the nucleosome (and identify its functions)
A nucleosome consists of DNA and histone proteins
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• DNA is wrapped around an octamer of histone proteins
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• Nucleosomes are linked by an interconnecting H1 histone
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Nucleosomes serve two key functions:
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• They help to supercoil DNA (improves packaging)
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• They help to regulate transcription
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List five examples of non-coding DNA
Satellite DNA (e.g. short tandem repeats)
S ………………………………………………………………………………………………………………………
Telomeres (the terminal sections of chromosomes)
T ………………………………………………………………………………………………………………………
Introns (non-coding sequences within genes)
I ………………………………………………………………………………………………………………………
Non-coding genes (e.g. genes for tRNA or rRNA)
N ………………………………………………………………………………………………………………………
Gene regulatory sequences (e.g. promoters, enhancers, silencers)
G ………………………………………………………………………………………………………………………
Explain the role of tandem repeats in DNA profiling
Short tandem repeats (STRs) are short repeating segments within satellite DNA
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The number of repeats for a particular loci will differ between individuals
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The STRs can be excised and separated on a gel to create a distinct DNA profile of a given individual
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(the more STR loci included in the profile, the more unique the DNA profile will be for the individual)
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Use the diagram below to outline the methodology and conclusions of the Hershey-Chase experiment
Hershey and Chase demonstrated that DNA was the genetic material by using radioactively labelled viruses
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• Viruses were prepared with radioactive phosphorus (labels DNA) or radioactive sulphur (labels protein)
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• Viruses then infected bacteria, before the bacteria and virus were separated via centrifugation
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(bacteria is heavier and forms a pellet, while the smaller virus remains in the supernatant)
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• When radioactive sulphur was used, radioactivity was detected in supernatant (not transferred to bacteria)
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• When radioactive phosphorus was used, radioactivity was detected in pellet (WAS transferred to bacteria)
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DNA Replication
State the direction of DNA replication
Replication occurs in a 5’ - 3’ direction (on the newly synthesised strand)
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State, with the aid of the diagram, the role of the following components of the DNA replication process
Unwinds and separates double stranded DNA (breaks H bonds between the base pairs)
Helicase: …………………………………………………………………………………………………………………………………………
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Relieves torsional strain created by helicase action (i.e. prevents supercoiling)
DNA Gyrase: ……………………………………………………………………………………………………………………………………
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Prevents DNA strands from re-annealing (SSB proteins will be displaced by DNA pol III)
SSB Proteins: …………………………………………………………………………………………………………………………………..
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Lays down a short RNA primer to provide an initiation point for polymerisation
DNA Primase: ………………………………………………………………………………………………………………………………….
(DNA pol III can only add nucleotides to the 3’-end of an existing nucleotide chain)
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Extends the nucleotide chain from the primer
DNA Pol III: ……………………………………………………………………………………………………………………………………..
(dNTPs align opposite complementary bases and DNA pol III covalently joins them together)
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Removes and replaces RNA primers with DNA nucleotides
DNA Pol I: ……………………………………………………………………………………………………………………………………….
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Covalently joins Okazaki fragments together (on lagging strand)
DNA Ligase: …………………………………………………………………………………………………………………………………….
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Outline the difference between leading and lagging strands as they relate to Okazaki fragments
DNA strands are antiparallel, so DNA pol III moves in opposite directions on the two strands
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On the leading strand, DNA pol III moves in the same direction as helicase - so synthesis is continuous
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On the lagging strand, DNA pol III moves in the opposite direction to helicase – synthesis is discontinuous
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The fragments generated on the lagging strand are called Okazaki fragments
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Describe the role of deoxynucleoside triphosphates (dNTPs) in the replication process
Deoxynucleoside triphosphates (dNTPs) align opposite their complementary base partner
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DNA pol III cleaves two of the phosphates and uses the energy to form a covalent phosphodiester bond
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In this way, DNA pol III synthesises a new DNA strand
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Explain, with the aid of the following diagram, how dideoxynucleotides are used in DNA sequencing
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form a phosphodiester bond
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This means the inclusion of a ddNTP will terminate the extension of a DNA sequence at that point
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Four PCR cycles are set up, each with a different ddNTP (ddA, ddT, ddG or ddC) and a stock of normal bases
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Each time the ddNTP is incorporated the sequence stops, generating fragments
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When these fragments are separated and then ordered according to length, the DNA sequence is discerned
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This process can be automated by using fluorescently labelled ddNTPs that can be detected by machine
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