topic 7 - Nucleic Acid (HL) Flashcards
How did Hershey and Chase provide evidence that DNA is the genetic material of the cell and not protein?
Known that viruses insert their genetic material into cells
radioactive viruses grown –> allowed to infect a bacterium (E. colu)
- Viruses grown in 35S had radioactive proteins (no DNA) but did NOT transfer this radioactivity to bacterium (remained in supernatant)
- Viruses grown in 32P had radioactive DNA (no protein) and DID transfer this radioactivity to infected bacterium (found in pellet)
virus & bacteria separated via centrifugation. Showed the above results
This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria
Who used X-ray diffraction to elucidate DNA structure?
Franklin and Wilkins
What is X-ray Diffraction?
- X-rays will diffract when targeted at crystallised DNA molecules
- The scattering pattern created can be used to determine structure
From the scattering patter created by X-Ray Diffraction, what are the properties that can be deduced?
- Composition: DNA is a double-stranded molecule
- Orientation: The bases face inwards and the phosphates face out
- Shape: DNA forms a double helix (10 bases per twist)
True or False?
In eukaryotes, DNA associates with eight histone proteins to form a nucleosome
TRUE
How does nucleosomes help to supercoil DNA?
- Makes DNA compact (better storage)
- Prevents DNA damage (less exposed)
- Assists in cell division (more mobility)
- Involved in transcriptional regulation
What are the regions of DNA that do not code for protein?
Think: STING
- Satellite DNA (tandem repeats)
- Telomeres (chromosome ends)
- Introns (non-coding sequences)
- Non-coding RNA genes
- Gene regulatory sequences
What are used in DNA profiling?
Tandem repeats
Describe how DNA –> Chromosome
DNA is bound with histone proteins to form nucleosomes that are then linked together to form strings of chromatosomes
These coil to form solenoids, which condense into 30 nm fibres, before being compressed and folded into chromatin
Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope
What is the role of helicase in DNA replication?
- Helicase separates the DNA strands to form a replication fork (breaks the hydrogen bonds between complementary base pairs)
- Single stranded binding proteins prevent strands re-annealing
What is the role of DNA Gyrase in DNA replication?
- DNA gyrase reduces the torsional strain created by helicase
- It prevents the DNA from supercoiling as it is being unwound
What is the role of DNA primase in DNA replication?
• DNA primase generates a short RNA primer on each strand
• PrimersprovideaninitiationpointforDNApolymeraseIII
(DNA pol III can only add nucleotides to 3’-end of a primer)
What is the role of DNA Polymerase III in DNA replication?
- Free nucleotides (dNTPs) line up opposite complementary bases
- DNA polymerase III covalently joins free nucleotides together
What is the role of Okazaki Fragments in DNA replication?
- DNA strands are antiparallel, so replication occurs bidirectionally (replication always occurs in a 5’ → 3’ direction on each strand)
- Synthesisiscontinuousontheleadingstrand(towardsfork) and is discontinuous on the lagging strand (away from fork)
- Discontinuous segments are called Okazaki fragments
What is the role of DNA Polymerase I in DNA replication?
• DNA pol I removes RNA primers and replaces them with DNA
What is the role of DNA ligase in DNA replication?
• DNA ligase covalently joins the Okazaki fragments together
Briefly describe the DNA replication process
- origins in DNA (eukaryotes have multiple, prokaryotes only have ONE)
- helicase unwinds DNA
2a. single-stranded binding (SSB) proteins keep DNA separated
2b. topiosomerase prevents supercoiling - Primase makes RNA primers
- DNA polymerase III builds the new strand in ONLY 5’ –> 3’ direction; proof reading ability prevents mistakes
- DNA polymerase I removes primer
- ligase seals Okazaki fragments on lagging strand tgth
- new strand
What is DNA sequencing?
Sequencing is a technique by which the nucleotide base order of a DNA sequence is elucidated (typically via Sanger method)
Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing (use of chain-terminating dideoxynucleotides)
Briefly describe the mechanism of DNA sequencing. What is the Sanger method?
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form covalent bonds (they terminate replication)
SANGER METHOD:
Four PCR mixtures are prepared – each with stocks of normal bases and one dideoxynucleotide (ddA, ddT, ddG, ddC)
Whenever the dideoxynucleotide is randomly incorporated, the DNA sequence is terminated at that base position
Because a complete PCR cycle generates millions of sequences, every base position is likely to have been terminated
These sequences are separated by gel electrophoresis to determine base sequence
(according to ascending sequence length)
Automated machines can determine the sequence quickly if fluorescent labeling of the dideoxynucleotides has occurred
True or False?
A gene is a sequence of DNA which is transcribed into RNA • Most genes encode proteins, but some do not (e.g. tRNA)
True
What are the 3 main sections of a gene sequence?
- Promoter (transcription initiation site)
- Coding sequence (the region transcribed)
- Terminator (transcription termination site)
True or False
As DNA is double stranded, only one strand is transcribed
• The antisense strand is not transcribed into RNA
• The sense strand is transcribed into RNA
FALSE
As DNA is double stranded, only one strand is transcribed
• The antisense strand is transcribed into RNA
• The sense strand is not transcribed into RNA