Nucleic Acid Analysis Flashcards
Nucleases
- sugar specificity
- can be specific for single or double stranded DNA
- differ by where in the phosphodiester bond they cut
- leave either the 5’ phosphate + 3’ hydroxyl or 5’ hydroxyl + 3’ phosphate
Endonucleases
cleave site within molecule
Exonucleases
remove single nucleotide from ends
- cut to leave phosphate on 5’ side with OH on 3’ side
- cleave polynucleotides hydrolytically at internal sites
Restriction Enzymes
endonucleases cutting both phosphate backbones to cleave target in two
Mirror like palindrome
sequence reads the same forwards and backwards on a ssDNA
Inverted repeat palindrome
sequence of top strand read left to right is the same as the sequence of the bottom strand read right to left
Methylation of DNA
- modification of DNA via methylation in restriction enzyme recognition site protects DNA from degradation
- C5 or N4 position of a cytosine
- N6 of adenine
- changes affect major groove of DNA
Replication and methylation
- only non methylated bases are used by DNA polymerase
- after replication, DNA will be hemi-methylated
- hemi-methylated DNA is the best substrate for methylases so it quickly becomes fully methylated before the next round of replication
Methylation and foreign DNA
- foreign unmethylated DNA coming into the cell is degraded, as this is more efficient that methylating the DNA
- protects genomic DNA/distinguishes foreign
Type I Endonuclease
- asymmetric discontinous recognition site
- methylate recognition site but cleave remotely
- multi subunit protein complex usually containing 2 restriction endonuclease subunits, 2 methyltransferase subunits, and 1 specificity subunit
- ATP needed for cleavage
- loop DNA so it binds to both cutting and recognition site
Type II Endonuclease
- symmetric recognition
- cleave/methylate close to/within recognition site
- REase usually acts independently of ATase
- acts as monomer, dimer or tetramer, ATP not required for cleavage
Type III Endonuclease
- complex with both REase and MTase subunits
- ATP needed to cleave
Restriction enzyme ends
- sticky ends: overhangs
eg. type II enzymes produce 5’ or 3’ overhangs
more useful as H bonding facilitates permanent linkages - blunt ends: are more difficult to connect, as they need ligase
Isoschizomers
enzymes with the same recognition site but not necessarily the same cleavage site or methylation sensitivity
Gel Electrophoresis
migration of charged molecules when under the influence of an electric field
- at eq., there is no net force on molecules
- mobility dependent on net charge and molecular dimensions
- used to separate molecules into components based on size and charge
Electrophoretic Mobility
v/E or q/f
frictional force = force exerted by electric fiel on particles
f x v = q x E
E = magnitude of electrical field
Agarose Gel Electrophoresis
agarose: linear polymer extracted from seaweed
- horizontal movement
- negative DNA moves to positive end
mobility decreases with increased gel concentration
Factors affecting DNA migration
- conformation of DNA
- agarose gel concentration
- size of DNA
- electrophoresis conditions
Plasmid DNA
Form 1: supercoiled covalently closed circle
Form 2: nicked relaxed covalently closed circle
Form 3: linear dsDNA fragment
Determination of the size of linear fragments
- calibration curve constructed using markers of known size
- semi log plot
- dn: distance of migration of fragment of known size
- graph log of size against distance of migration
- dx (distance of unknown fragment) on x axis, go up until you hit the line then go across to find size
PAGE electrophoresis
polyacrylamide gel electrophoresis
- vertical slab gels
Denatured Electrophoresis
- SDS detergent stretches protein chain and sticks onto the protein
- masks protein to give it an overall negative charge
- longer protein = more negative charge so moves slower
- constant charge/mass ratio allows molecular masses to be estimated
Native Electrophoresis
Non denaturing PAGE
-WHAT GOES HERE IDK
DNA Sequencing
- production of a series of ssDNA molecule with common 5’ end
- identity of 3’ end base known
- molecules separated using PAGE in presence of urea
Maxem and Gilbert Method
- separate duplex DNA to ssDNA
- addition of radioisotope (32P) at 5’ end
- chemical cleavage at 5’ end of specific nucleotides
- obtain sequencing latter by gel electrophoresis
4 outcomes: G, G+A, T+C, C
Sanger Method
- uses fluorescently labelled dideoxynucleotides to stop replication
- partial copies of template synthesized and separated by size
- sequence determined based on size of molecule and labelled ddNTP
Next Gen Sequencing
- fragment DNA and attach to solid surface
- add end adaptors
- primers complementary to adaptor
- DNA extension to give sequence
- fluoresence labelling
- large number of samples sequenced at the same time
