FHMP 002 DNA structure and replication Flashcards
What is the structure of DNA?
- composed of polynucleotides
- have a phosphate group, deoxyribose sugar, and a nitrogen-containing bases (adenine, thymine, cytosine and guanine)
- Has a doubled stranded helix that is formed by hydrogen bonds between polynucleotides
- made of deoxyribonucleoside triphophates (dNTP)
whats a nucleotide and a nucleoside?
- nucleotide = base + sugar + triphosphate
- nucleoside = base + sugar
what are the complementary base pairing rules?
- Adenine pairs with Thymine (or ur and Cytosine pairs with Guanine
- 2 bonds between A and T and 3 bonds between C and G
- Purine binds with pyrimidine
What are purines and pyrimidines?
Purines: Adenine and Guanine (2 big rings)
Pyrimidines: Thymine and Cytosine and Uracil ( 1 small ring)
What is DNA replication?
the process of copying DNA
- semi conservative
Describe the DNA replication process
- Enzymes gyrase unwind and DNA A and DNA B (helicase) unzips the double helix and hydrogen bonds are broken. Single-stranded binding (SSB) proteins keep strands separate. Bases are exposed
- DNA/RNA primers are added by interacting with the SBBs
- the leading strand is synthesised continuously from 3’ to 5’ and the lagging strand is synthesised in fragments in the Okazaki method in the 5’ to 3’ direction
- Free nucleotides pair with exposed bases, & DNA polymerases bond together and form complementary strands and DNA ligase seals together the fragments to make a continuous strand
- 2 identical double-stranded DNA molecules result from replication. DNA replication is semiconservative as the new molecule has 1 old strand and 1 new.
what are the functions of DNA polymerase and exonuclease?
- 5-3 DNA polymerase = synthesising DNA (co factor = Mg ions)
- 5-3 exonuclease = primer removal
- 3-5 exonuclease =for proof reading (cleaves incorrect nucleotides)
What is DNA sequencing?
The process of determining the precise order of nucleotides within a DNA molecule
what do you need for DNA sequencing?
- normal nucleotides (dNTPs) = deoxyribonucleotide triphophates
- terminal nucelotides (ddNTPs) = dideoxyribonucleotide triphophate ( lack OH group)
- DNA template
- DNA primer
- DNA polymerase
- possible radioactive or florescent labelling
describe the process of DNA sequencing
- DNA samples are seperated into 4 reactions
- each reaction contains all 4 normal dNTPs (A,T,C,G), primer, and polymerase and 1 of the 4 ddNTPs is added
- the ddNTPs are labelled with radioactive/fluorescent tags
- DNA replication occurs and the ddNTPs are added to the DNA sequences, but cause chain termination at the different bases in each reaction, creating lots of fragments of DNA
- electrophoresis is then used to separate the strands by size
- this will be run through a computer/data logger and identify where each base is along the chain by identifying where the tagged ddNTPs are
What is PCR?
Polymerase Chain Reaction; used to amplify DNA
What do you need for PCR?
- taq polymerase
- DNA template
- deoxynulceotides triphosphates ATCG
- primers
- cofactors (Mg for taq pol)
- buffer solution
- thermal cycler
describe the process of PCR
- Denaturation = DNA is heated to 95°C causing it to denature which separates the strands.
- Annealing = It is then cooled to 55°C allowing complementary primers to anneal to specific target sequences.
- Elongation = Finally it is heated to 72°C allowing heat-tolerant Taq polymerase to synthesise new strands from free DNA nucleotides in 5’ to 3’ direction
- Many repeated thermal cycles (often 30) allow billions of copies of the target sequence to be produced.
What is next generation sequencing?
High-throughput sequencing technologies that parallelise the sequencing process, producing thousands or millions of sequences at once e.g. illumina sequencing
What is illumina sequencing?
Sequencing using the dideoxy chain termination method, but attaching a removable fluorescent molecule to each nucleotide, with a different colour for each base.
Instead of using ddNTPs, a chemical group that blocks elongation by DNA polymerase is added to the dNTP, which can be removed chemically.
