DNA Manipulation and Replication Flashcards
RNA Structure
Consists of a ribose sugar and a phosphate group attached to the 5’ carbon. A nucleic acid base is attached to the 1’ carbon. RNA bases are AUCG.
DNA Structure
Consists of a ribose sugar and a phosphate group attached to the 5’ carbon and a nucleic acid base attached to the 1’ carbon. DNA bases are ATCG.
Purines
Consists of the bases G and A, which are two cyclic molecules linked together
Pyrimidines
Consists of the bases C, T and U, which are single cyclic molecules.
Phosphodiester bond
Bond between a phosphate group and the ribose sugars on either side, links two successive sugar molecules together. (Made up of two esther linkages).
Esther Linkage
The bond between the O of the phosphate group and the carbons of the sugar
Nucleotides can only be added to the
3’ end. Because the OH on the 3’ end can attack the high energy phosphate bond of the incoming nucleotide - causes synthesis
Major groove
A wider, thicker section of double helix
Minor Groove
A smaller, thinner section of double helix
There are _____ bases per complete turn
10
Bases are _______ and the backbone is ________
hydrophobic, hydrophilic
Primary Structure of DNA
Order of bases in the DNA strand
Watson, Crick, Wilkins
Determined the structure of DNA. Two DNA strands line up in an antiparallel manner, and twist to form a double helix
Secondary Structure of DNA
Stabilized by complimentary base pairing and by ‘Base stacking’.
Rosalind Franklin
Crystalography of DNA.
Chargaff’s Rule
Found that the amount of guanine and is the same as the amount of cytosine, and the amount of adenine is the same as the amount of thymine. Decided they are bonded together. Through radioactive labeling.
Pyrophosphate
2 phosphate groups removed from ATP releasing a lot of energy. Coupled with adding incoming nucleotides to the chain
Forces stabilizing DNA
H-bonds
Hydrophobic, hydrophilic interactions
‘Base stacking’, or Van der Waals forces
Electrostatic interactions between bases
Replication
Two strands of the parental duplex separate at the replication fork. Each parental strand serves as a template for the synthesis of a new daughter strand
DNA replication is
Semiconservative
Miselson and Staal
Used radioactive material to label and replicated DNA. Two bands were shown which proved that the process was semiconservative.
Leading Strand
Can be replicated continuously in the 5’ to 3’ direction
Lagging Strand
Is replicated in small segments discontinuously in the 5’ to 3’ direction
Okasaki Fragments
Short segments produced on the lagging strand
RNA Primase
Lays down primers to start replication, because DNA polymerase needs a free OH group to lay down nucleotides. The primers are removed by a different DNA polymerase
Ligase
Ligates the Okasaki fragments together after primers have been removed.
Helicase
Unwinds the parental DNA strands
Topiosomerase II
Relieves the stress of unwinding - cuts strands of DNA, unwinds it, and sticks it back together
Single-strand binding proteins
Stabilizes single strands of DNA, works next to helicase, protects the DNA when its unzipped as single strands
DNA Polymerases
There are five, DNA polymerase I and III are the most important. I fills in gaps left by RNA primers and is involved with DNA repair, and III is the main player in replication and has proof-reading abilities. The rest are involved in proof-reading and repair.
Replication bubble
Forms around the origin of replication, the replication fork proceeds in both directions from this area
Origin of Replication
Eukaryotes have many, and they fuse together when they meet. Prokaryotes have one.
Circular DNA
Still has a leading and lagging strand, replication starts at the origin and moves in both directions
Telomerase
Contains an RNA template that allows the shortened 3’ end of the template strand to be restored by the addition of more telomere repeats. Present mostly in gametes and stem cells.
Telomere
TAG repeating sequences on somatic cells. Needed because the lagging strand has a small section that goes unreplicated in eukaryotes.
PCR
Polymerase Chain Reaction. Has three stages, denaturation, annealing and extension. Kary Mullis.
Denaturation (PCR)
Solution containing DNA is heated to separate the DNA strands. Intention is to make large amounts of DNA
Annealing
Solution is cooled, two primers anneal to their complimentary sequence on the strands of the template duplex
Extension
DNA polymerase synthesizes new DNA strands by extending primers in a 5’ to 3’ direction
Gel Electrophoresis
DNA molecules are separated by size. Larger molecules move more slowly. DNA moves from the minus end to the plus end
Restriction enzymes
Cut DNA in set places, after identifying a specific 6 base sequences. Sticky end is when one end of the DNA cut is longer than the other. Blunt end is the opposite.
Restriction Fragments
Pieces of bacterial DNA cut by restriction enzymes
Southern Blot
DNA fragments are denatured and then transferred (blotted) onto filter paper. Then placed in a bag containing solution with a single-stranded labeled probe, which sticks to complimentary fragments by hybridization. Filter paper is then exposed to X-ray film. Purpose is to verify that a DNA fragment is what one thinks it is.
Sanger Sequencing
Template strand is unknown, purpose is to determine nucleotide sequence. Creation of a daughter strand is stopped when a terminator is incorporated at the 3’ end. Each dideoxynucleotide is labeled with a different florescent dye, and we can find the sequence of daughter strand. Run through gel electro and a detector picks up on each dye as it exits.
Recombinant DNA
Donor DNA and vector DNA are both cleaved with the same restriction enzyme, and joined together with DNA ligase. Can be inserted into bacterial population and uptaken through transformation.
Gene Editing
Guide RNA binds with Cas9 protein and brings it to the target DNA and the target is cleaved. An exonuclease widens the gap in the target DNA. Editing template is used to repair this gap. Result is edited DNA.
Dideoxynucleotide Terminator or Chain Terminator
Lacks an OH group that is needed to add additional nucleotides to the 3’ end. Terminates the creation of a DNA chain.
DNA is read in the
3-5 direction
DNA is replicated in the
5-3 direction
Midochondria and Chloroplasts have…
Circular DNA
