Nucleic Acid Analysis

Question 1

Nucleases

Answer 1

• 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

Question 2

Endonucleases

Answer 2

cleave site within molecule

Question 3

Exonucleases

Answer 3

remove single nucleotide from ends

• cut to leave phosphate on 5’ side with OH on 3’ side
• cleave polynucleotides hydrolytically at internal sites

Question 4

Restriction Enzymes

Answer 4

endonucleases cutting both phosphate backbones to cleave target in two

Question 5

Mirror like palindrome

Answer 5

sequence reads the same forwards and backwards on a ssDNA

Question 6

Inverted repeat palindrome

Answer 6

sequence of top strand read left to right is the same as the sequence of the bottom strand read right to left

Question 7

Methylation of DNA

Answer 7

• 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

Question 8

Replication and methylation

Answer 8

• 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

Question 9

Methylation and foreign DNA

Answer 9

• foreign unmethylated DNA coming into the cell is degraded, as this is more efficient that methylating the DNA
• protects genomic DNA/distinguishes foreign

Question 10

Type I Endonuclease

Answer 10

• 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

Question 11

Type II Endonuclease

Answer 11

• 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

Question 12

Type III Endonuclease

Answer 12

• complex with both REase and MTase subunits

- ATP needed to cleave

Question 13

Restriction enzyme ends

Answer 13

• 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

Question 14

Isoschizomers

Answer 14

enzymes with the same recognition site but not necessarily the same cleavage site or methylation sensitivity

Question 15

Gel Electrophoresis

Answer 15

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

Question 16

Electrophoretic Mobility

Answer 16

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

Question 17

Agarose Gel Electrophoresis

Answer 17

agarose: linear polymer extracted from seaweed
- horizontal movement
- negative DNA moves to positive end
mobility decreases with increased gel concentration

Question 18

Factors affecting DNA migration

Answer 18

1. conformation of DNA
2. agarose gel concentration
3. size of DNA
4. electrophoresis conditions

Question 19

Plasmid DNA

Answer 19

Form 1: supercoiled covalently closed circle
Form 2: nicked relaxed covalently closed circle
Form 3: linear dsDNA fragment

Question 20

Determination of the size of linear fragments

Answer 20

• 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

Question 21

PAGE electrophoresis

Answer 21

polyacrylamide gel electrophoresis

- vertical slab gels

Question 22

Denatured Electrophoresis

Answer 22

• 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

Question 23

Native Electrophoresis

Answer 23

Non denaturing PAGE

-WHAT GOES HERE IDK

Question 24

DNA Sequencing

Answer 24

1. production of a series of ssDNA molecule with common 5’ end
2. identity of 3’ end base known
3. molecules separated using PAGE in presence of urea

Question 25

Maxem and Gilbert Method

Answer 25

• 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

Question 26

Sanger Method

Answer 26

• 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

Question 27

Next Gen Sequencing

Answer 27

• 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