Chapter 3 Techniques

Where does DNA move towards in Gel Electrophoresis?

positive cathode

What determines DNA migration speed in a gel?

fragment size

What size of DNA fragments move faster through Gel Electrophoresis?

smaller

What % agarose dissolves smaller fragments?

large (2%)

What % agarose dissolves larger fragments?

small (0.7%)

What is restriction digest?

uses bacterial restriction enzymes to cut DNA at specific sequences

Why do bacteria have restriction enzymes?

defense against foreign DNA

What do restriction enzymes do?

cut DNA at specific recognition sequences

What two classes are restriction enzymes divided into?

specificity and ATP requirement

What enzyme cleaves at specific sequences (4-12 bp) and do NOT require ATP?

Type II restriction enzymes

What are sticky ends?

5’ or 3’ overhang that can base-pair with complementary DNA

What are blunt ends?

DNA cleaved at same nucleotide position on coding and non coding strands

What is restriction mapping used for?

finding DNA structure by fragment patterns

What is restriction mapping used for?

inherited genetic defects and forensic analysis

What are the two methods of digesting DNA?

single (one restriction enzyme) or double (two restriction enzymes)

What is Restriction Fragment Length Polymorphisms (RFLP)?

unique fragments produced from different DNA sources

What type of restriction enzyme: non-specific, needs ATP?

Type I

What type of restriction enzyme: specific, no ATP?

Type II

What type of restriction enzyme: specific, needs ATP?

Type III

What is a “six-cutter”?

enzyme that recognizes a 6-base sequence

What does Southern blot detect?

specific DNA fragments

Why denature DNA in Southern blot?

To allow single-stranded probe binding

What did Frederick Sanger develop?

chain-terminator method

What is the chain-terminator method based on?

DNA replication

Why was dideoxynucleotide (ddNTP) developed?

to terminate chain elongation

What causes chain termination?

lack of 3’ OH in ddNTPs

Why are 4 reactions used in classic Sanger sequencing?

Each contains a different ddNTP (A, T, C, G)

How is sequence read from gel?

bottom to top gives 5’ → 3’ sequence of new strand

What is the key improvement ovf automated DNA sequencing?

uses fluorescently labeled ddNTPs so only ONE reaction needed

What does each color on the chromatograph represent?

nucleotide type

What does each peak position on the chromatograph represent?

ddNTP at that position (sequence order)

What does each peak size on the chromatograph represent?

amount of ddNTP

How are the chromatograph peaks read?

left to right = 5’ to 3’

What is agarose used for?

large DNA

What is polyacrylamide used for?

small/high resolution DNA

Why is GC-rich DNA more stable?

has 3 hydrogen bonds + stronger base stacking

What do intercalating agents do?

insert between DNA bases and distort helix

What is the structure of intercalating agents?

aromatic macrocycles

What is the structure of aromatic macrocycles composed of?

flat, hydrophobic, fused heterocyclic rings

What is the function of Ethidium Bromide?

fluorescent DNA staining in gel electrophoresis

What is the function of Acridine Orange?

stain nucleic acids to study cell cycle

What is the function of Actinomycin D?

inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation

What is Actinomycin D?

antibiotic with anti-cancer effects

What is DNA denaturation?

Separation of double-stranded DNA into single strands

What is the hyperchromic effect?

exposed bases in denatured DNA results in an increase in absorbance (260 nm)

What is Tm?

Temperature where 50% of DNA is denatured

How can DNA be renatured?

by cooling

What does renaturation require?

reannealing, reassociation of the DNA strands into a double helix

How can reannealing occur?

the strands must realign so that their complementary bases are once again in register and the helix can be “zippered up”

What restriction endonuclease cuts once?

HindIII

What restriction endonuclease cuts twice?

BamHI

What type of sequence do restriction enzymes recognize?

palindromes

What does happens to bacterial DNA to protect it from restriction enzymes?

methylation