19 Nucleic Acid Analysis

Question 1

gel electrophoresis get types:

Answer 1

gel matrix mesh with pores, shape + size of DNA influences travel speed
Agarose: resolving power: 100s - few k bases long (medium sized fragments)

Polyacrylamide: used for small fragments 50 - 150 bases, higher rp - can differentiate differences of 1 base difference

Pulse field electrophoresis (uses normal agarose gel with modded procedure to have higher resolving power of long fragments): applies directional electric current in alternating directions, longer fragments take longer, rp: 100s kb - megabase range

Question 2

gel electrophoresis procedure

Answer 2

gel poured with wells + dyed Nucleic Acids, power supply and electrodes on both ends causes -ve charged DNA backbone to move from one end to the other.

Question 3

purpose of marker ladders (test bands running up the side)

Answer 3

tells you rough length of fragment if its band matched up with a marker of a known length

Question 4

Adding Formamide (chemical) to nucleotides + urea to polyacrylamide gels

Answer 4

stabilises single stranded RNA/DNA to stop it folding and binding with itself
= allows nucleic acids to separate based on size alone (not shape)
= denaturing condition
(urea: same role but added in polyacrylamide gel)

Question 5

Southern Blotting

Answer 5

Used to check definitively for a certain fragment

• Agarose gel used, Nitrocellulose filter added on top, salt solution on top of that = nucleotides transfer to filter through capillary action.
• filter then exposed to probe: radioactively labelled Oligonucleotide molecules which are complementary to the gene whose presence you are checking for. unbound probes washed away, if gene is present, the bound probe will stick.
• filter with bound probe exposed to radioactive ray = radioactively labelled gene will show up

Question 6

Oligonucleotides

Answer 6

Oligonucleotides, or oligos, are short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology and synthetic biology application - can be synthesised to have perfect complimentarily to specific target gene

Question 7

Northern Blotting

Answer 7

same as southern but uses RNA
extra step first to purify coding mRNA from all extracted cell RNA: oligoT dynabeads have TTTTTT chains attached to magnetic bead, binds to AAAAA (poly-A tail) on all mRNA to catch them, washes rest away

same radioactively labelled probe to detect gene present - application , can check for presence of a gene in mRNA (showing DNA expression) in different tissue cells.

Question 8

EMSA electrophoresis

Answer 8

used for testing for whether proteins bind to a DNA sequence using native (non-denaturing poluacrylamide gel electrophoresis - PAGE)
eg. transcription facators (methylation. acetylation),
DNA ran next to DNA + protein, slower migration if protein binds

Question 9

SSCP electrophoresis

Answer 9

uses native conditions
tests for gene mutation without need for sequencing
known normal ‘non-mutant’ DNA single stranded DNA ran alongside suspected mutant DNA- will run at different speed due to structure (shape) shift from mutated native binding
heat + rapidly cool to separate double stranded DNA

Question 10

why is DNA amplified for electrophoresis?

Answer 10

using PCR to clone DNA

Many fragments of the same length must accumulate to form a strong visible band

Question 11

Process of Sanger sequencing

Answer 11

• to sequence a genome/ find base sequence of DNA
• PCR used to amplify
• uses ddNTPs to make fragments - so many copies that there will be a strand ending in a ddNTP in every base
• ddNTPs are fluorescently labelled to glow different colours for different bases.
• ran on PAG and read from the bottom of gel up in opposite bases (1st nucleotide = 1 base long so travels furthest, second = 2nd longest etc.)
• sometimes displayed as an electropherogram

Question 12

what is/ how to read an electropherogram

Answer 12

a graph made to display raw results from Sanger sequencing

- to read, look at each peak’s colour corresponding to each base in order from left to right (complimentary bases)

Question 13

Next generation sequencing (NGS)

Answer 13

• illumina sequencing

Question 14

illumina sequencing

Answer 14

• reversible fluorescent terminators (‘reversible dye terminator), bound to a glass slide (PCR on a slide)
• faster than sanger, used for large genomes
• fragment whole genome into many fragments (ddNTP?)
• glass slide with primers attached and attached version of fragment to replicate, bridge reaction to copy complimentary strands, denatured to separate, repeat
• laser used to excite each fluorophore molecule before being cleaved off and reversible terminator in place of OH group on sugar