Topic 7 Flashcards
Describe the Hersey and Chase experiment
- provided evidence that DNA is the genetic material
- T2 bacteriophage were grown in two isotopic mediums
- viruses were grown in radioactive sulfur with radiolabelled proteins as sulfur is present in proteins but not DNA
- and viruses were also grown in radioactive phosphorous with radiolabelled DNA ( phosphorous is present in DNA)
- the viruses then infected bacteria and the two were separated by centrifugation
- The bacteria was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)
Describe x ray diffraction
- Used to investigate the structure of DNA
- DNA was purified and then fibres were stretched in a thin glass tube
- The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom
- The scattering pattern of the X-ray was recorded
What evidence did xray diffraction provide for the structure of DNA?
- double helix structure
- double stranded
How does the structure of DNA suggest a mechanism for DNA replication?
- Chargaff had also demonstrated that DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T) .Shows that nitrogenous bases are paired within the double helix and the two strands must run in anti-parallel directions
- Franklin’s x-ray diffraction experiments demonstrated that the DNA helix with P on the outside. The P form an outer backbone and nitrogenous bases are packaged within the interior
- DNA replication occurs by complimentary base pairing and is bidirectional
Describe the process of DNA replication
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs creating a replication fork of two strands running in antiparallel directions
- DNA gyrase relieves the tension in the strand.
- DNA primase generates a short RNA primer providing an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one.
- Free nucleotides align opposite their complementary base partners and DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments). - As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides - DNA ligase joins the Okazaki fragments together to form a continuous strand by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
What can you use to stop DNA replication in preparation of samples for base sequencing?
dideoxyribonucleic acid
What is DNA sequencing?
Making the base order of a nucleotide sequences clearer
What about dideoxynucleotides make them stop DNA replication?
-Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond which prevent further elongation of a nucleotide chain and terminate replication.
Describe Sanger’s method for DNA sequencing
- Four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide
- Each PCR mix should generate all the possible terminating fragments for that particular base
- When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
- If a primer is included in each mix, the fragments can be detected by automated sequencing machines
If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand
What are the other important functions of DNA except for
coding for proteins?
- Telomeres ( regions of repetitive DNA at the end of a chromosome that protects chromosomal deteriation)
- gene regulation ( sequences that are involved in translation/transcription like promoters)
- Non coding RNA genes ( Codes for RNA molecules that are not translated)
- Tandem repeats of DNA
What are tandem repeats used for?
DNA profilling
Describe the process of DNA profilling using tandem repeats
- Within the non-coding regions of an individual’s genome there are short tandem repeats
- Tandem repeats can be removed using restriction enzymes and then separated with gel electrophoresis for comparison
- As individuals will have different numbers of repeats at a given satellite DNA locus so will generate unique DNA profiles
- Longer repeats will generate larger fragments, while shorter repeats will generate smaller fragments
Nuclesomes
What are the three main components of a gene?
-promoter, coding sequence, terminator
What is the promoter an example of?
Non coding DNA with a function
Name some facts about the promoter
- The non-coding sequence responsible for the initiation of transcription
- Binding site for RNA polymerase (the enzyme responsible for transcription). Binding is controlled by transcription factors.
These transcription factors bind to either near or away from the promoter