D1.1 DNA Replication Flashcards
Reasons for DNA replication
- cell division
- reproduction
semi coneservative replication
Type of DNA replication in which each double strand of DNA contains one strand of the original DNA and one strand of newly synthesised DNA
activated nucleotide
has 3 phosphate groups
Semi conservative replication outlines
- Hydrogen bonds between bases break
- free nucleotides are present in the nucleus
- free neucleotides pair up with complementary exposed bases
- new strand is linked together
- now two DNA molecules => each one old and one new strand
evidence for semi conservative replication
- cultured e.colibacteria in the presence of heavy nitrogen isotope (15N)
- bacterial culture transferred to fresh medium = nitrogen replaced with 14N
- after one generation/division of bacteria the resulting DNA strand consisted of one strand 14N and one strand 15N
Gel electrophoresis
molecules are seperated by their size and amount of charge
uses an electrical current to move molecules through a semisolid medium
Facts about gel electrophoresis
- DNA/RNA has negative charge -> moves to positive electrode
- 250-30000 bp in length fragments
- samples with fragments are loaded into small wells
Key points of gel electrophoresis
- DNA must be colored with ethidium bromide
- seperates by charge and size
- consistency of gel allows seperation of the DNA fragements by size
- DNA must travel through spaces between polymers
- smaller pieces can slip through easily => travel further
PCR Test stages
- Denaturing to seperate DNA strands
primer, DNA fragments, polymerase and nucleotoides added to thermocycler (95 degrees)
- Annealing of primers
primers join to their complementary basses at 55-68 degrees
- synthesis of DNA
temperature increased to 72 degrees; optimum of DNA polymerase= Taq attached to nucelotides of seperated DNA
=> repeats multiple times to amplify
Advantages of PCR
- very rapid
- does not require living cells
PCR
polymerase chai. reaction in which DNA is amplified for further processing or study
applications of PCR
- Tissue Typing = donor & recepient can be matched to reduce rejection
- Detection of oncogenes = detect type of mutual leading to cancer
- detecting mutations = genetic diseases
- identifying viral infections = verify the type
- monitoring spread of infectious disease
- forensiuc science = identify criminals
- research = extinct organisms
Things needed for PCR
- Taq polymerase (DNA polymerase)
- small DNA sample
- thermocycler
- primers
- nucleotides
DNA profiling stages
- extract DNA from sample
- digest sample using resitriction endonuclease
- seperate DNA fragments using electrophoresis
- DNA seperated into single strand susing alkaline solution
- DNA fragments transferred to nylon membrane by southern blotting
- Hybridisation of DNA probes added to label fragments
- development (placed into x ray)
Lagging strand
DNA strand that is synthesised discontinuously in short fragments
Directionality of DNA replication
5 -> 3
* phosphate group on the 5th C of the pentose sugar covelently bonds to OH on 3rd C
* DNA polymerase III adds the 5 end of a DNA nucleotide to 3 end
DNA
Leading strand
DNA strand that is synthesised continuously in the 5 to 3 direction
How the lagging strand operates
- DNA polymerase III replicates new strand in sections
- repeatedly move further along the strand to continue replication
- sections of disconnected DNA = okazaki fragments
- DNA ligase joins Okazaki Fragments together
Gyrase (Topoisomerase II)
moves ahead of helicases, relieving tension created by the unwinding and unzipping of DNA
Helicase
unwinds & unzips the DNA molecule by breaking H bonds holding compl. bases together
Ligase
catalyses formation of phosphodiester bonds between okazaki fragments
* works primarily on lagging strand
DNA polymerase I
removes RNA nucelotides of the primers and replaces them with correct DNA nucelotides
* works primarily on lagging strand
DNA primase
attracts small RNA primers, made up of RNA nucleotides to the template strand = allows DNA polymerase III to attach and begin assembling free nucleotides = new DNA strand
DNA polymerase III
assembles new strand of DNA by placing new nucleotides in the correct sequence according to the base sequence of the template strand and comp. base pairing rule
* operates in 5 to 3
* proofreads
* works continuously on leading strand
* works dicontinuously on lagging strand
DNA proofreading
DNA polyemerase III proofreads newly built DNA = removes incorrectly placed nucleotides & replaces with correct one
DNA proofreading steps
- DNA polymerase III adds incorrect nucelotide while building new DNA
- DNA polymerase III identifies the error as it does not match the base pair
- DNA polymerase III removes incorrect nucleotide
- DNA polymerase III adds correct nucleotide
Which enzymes are needed for the leading strand
DNA polymerase, DNA Primase, one RNA primer
What enzymes are needed for the lagging strand
DNA polymerase I &III, DNA primase, RNA primers, DNA Ligase
How DNA proofreading works
- DNA polymerase III has a proofreading function = 3’ to 5’ exonuclease activity.
- During DNA replication, if an incorrect nucleotide is incorporated; enzyme detects mismatch ( improper hydrogen bonding)
- exonuclease activity removes the incorrect nucleotide, = polymerase to insert the correct one
- crucial for maintaining replication fidelity, reducing the error rate and ensuring the accurate transmission of genetic information.
DNA profiling outlined
- take DNA sample
- PCR to amplify using Taq polymerase
- do tandem repeats
- use gel electrophoresis to seperate DNA
- seperation according to fragment length
- name application; forensic, crime, paternity
