Ch. 9 & Ch. 13 - Nucleic Acids & Nucleic Acid Biotech Techniques

pyrimidines

compounds that contain a 6-membered ring

• C, U, T

purines

compounds that contain a 6-membered ring connected to 5-membered ring

• A, G

when does DNA denature and renature?

Is C + G stronger than A + T?

yes, 3 H-bonds vs. 2

circular DNA

double-stranded DNA where 5’

negative supercoils

circular DNA w/ fewer than normal # of turns of the helix

positive supercoils

circular DNA w/ more than normal # of turns of the helix

topoisomerases

enzymes that relax supercoiling in closed circular DNA

• 2 types:

  • Class I – cut the phosphodiester backbone of 1 strand, pass the other end through, & reseal

  • Class II -

DNA gyrase

Chromatin

complex of DNA & protein found in eukaryotic nuclei

Histones

basic proteins complexed to eukaryotic DNA

• 5 main types:

  • H1, H2A, H2B, H3, H4

• rich in basic a.a residues: K & R

nucleosome (individual beads of the chromatin)

globular structure in which DNA is wrapped around an aggregate of histone molecules

• each core has 8 histones: H2A, H2B, H3, & H4 (2 of each)

spacer regions

string portions of chromatin w/o nucleosome

hyperchromicity

melting

heat denaturation of DNA

T_M

• melting temperature

• midpoint of melting curve

• T_M ⬆ when C–G % is higher

• T_M ⬆ when pH ⬇ & vice versa

• T_M ⬆ when DNA strand

t(transfer)RNA

• single stranded polynucleotide chain btw 73 & 94 nucleotides long

• carries a.a at 3’ end

• intrachain H-bonding

r(ribosomal)RNA

• 2 subunits (1 larger than the other)

m(messenger)RNA

• initially larger precursor molecule–heterogeneous nuclear RNA (hnRNA)

• carries coded genetic info from DNA → ribosomes

• small amount in cells & short lived

• copies info from top strand of DNA (5’–3’)

sn(small nuclear)RNA

• 100–200 nucleotides long

• in nucleus of eukaryotic cells

snRNPs (type of snRNA)

Gel Electrophoresis

• separate molecules based on charge:size ratio

  • DNA already –

• electric current applied

• agarose

autoradiography

locating radioactively labelled substances by exposing them to photographic film

fluorescence

sensitive method for detection & ID of substances that absorb & re-emit light

• ethidium bromide (EtBr)

  • slips btw DNA bases

  • strong carcinogen

• SyBr Green & SyBr Gold newer options

nucleases

enzymes that hydrolyze nucleic acids

exonuclease

cleaves from ends of molecule

endonuclease

cleaves in middle of chain

restriction endonucleases

enzymes that hydrolyze double-stranded DNA at specific spots on opp. strands

• recognizes only specific palindromes

palindrome

msg that reads the same backward or forward

sticky ends

short, single-stranded stretches at ends of double-stranded DNA

• can join by H-bond btw complementary bases

• provides site where other DNA w/ sticky ends can be linked

DNA ligases

enzyme that join DNA strands tgt through phosphodiester bond formation

4 cutters, 6, cutters, 8 cutters

plasmids

DNA molecules that contain genes for antibiotic resistance & are used in cloning

selection process

allows bacteria that have been transformed to be identified & isolated

• plasmid has selectable marker for ID

ampicillin (Amp^r) & tetracycline (Tet^r)

antibiotics

Ori

origin of replication—DNA sequence that tells bacteria to replicate

multiple cloning sites (MCS)

region of bacterial plasmid that has short stretch of DNA w/ many restriction sites

why having 2 restriction enzymes is beneficial (2 different sticky ends)

• will not self-ligate bc not complementary

• insert will only go in 1 direction (guarantees directionality)

gene therapy

cells of specific tissues altered to alleviate symptoms of a disease

production of recombinant human insulin

1. insert DNA encoding A chain & B chain into separate plasmid expression vectors

2. transform E. coli w/ vectors & clone separately

3. add induce to start protein synthesis

4. purify & mix A and B chains tgt

expression vectors/plasmid

plasmids that

Polymerase Chain Reaction (PCR)

like cloning but no reliance of any organism such as bacteria

PCR steps

1. template/targeted sequence (must know the

2. primers (complementary to each template) added

   • 3’ ends inward toward desired

3. DNTPs added to build the DNA

4. Taq DNA polymerase added

   • comes from thermus aquaticus

5. heat to 95ºC (denature) & cool to 55–60ºC to anneal

how to figure out number of copies generated from PCR

2ⁿ; n = number of cycles