5500A Midterm I

Lysis Buffer Makeup

detergent, protease, EDTA and salt

Differential Lysis

incubation in hypotonic buffer lyses RBCs via osmosis

Tissue Sample Prep

Fresh/ Frozen samples must be dissociated via homogenization/ grinding/ sonication

Fixed/ Embedded Tissue must deparaffinize and rehydrate

cfDNA procedure

uses tubes to prevent lysis and release DNA, separate from protein and cell

Liquid DNA Isolation - Inorganic Reagents Used

detergents, EDTA, salt

fast, no fume hood, and high-quality DNA

Salting Out - Inorganic Liquid Extraction Steps

1. Lysis: cell membranes are lysed and proteins denatured by detergent (SDS)

2. Precipitation: proteins high salt low pH and DNA with ethanol

3. Resuspended: TE buffer or water

DNA Precipitation - OH and Salt Addition =

makes DNA less hydrophilic and easy to precipitate

DNA Precipitation - Lower Temp =

promote clumping = pellets

DNA Precipitation Isopropanol > Ethanol

makes DNA less soluble = precipitate faster at lower concentration but salt will too

1:1 ratio at room temp to reduce salt precipitation

FFPE Tissue

formalin-fixed paraffin-embedded tissue, results in variety of cross-linking between DNA and proteins

mRNA Isolation

done via oligo dT probes in column/ beads

mRNA Elution

low-salt and mild detergent to break hydrogen bonds between mRNA and column

UV Spectrophotometry micrograms/ mL

50 micrograms/ mL dsDNA

40 micrograms/ ml RNA

A260/280

measure of purity measuring protein contamination

A260/230

measure of purity measuring other contaminants (phenol, salt, carbohydrates)

Unsatisfactory A260/230 Consequence

salt carryover would impede elution of nucleic acid = poor yield

Good DNA OD260/280

1.6-2, >2 may be contaminated with RNA

Good RNA OD260/280

<1.6 too much protein or organic compounds

Fluorometry

utilizes fluorescent dyes that bind DNA/ RNA that increase when bound, requires negative control

Agarose Gel %

>5% or <0.3% are not practical for use

decided by the size of DNA as gel/ buffer will affect resolution

PAGE (polyacrylamide gel electrophoresis)

higher resolution capacity than agarose for small fragments

resolves 1bp difference in a 1 kb molecule

Tris Makeup

electrophoresis buffer, borate (salt), and EDTA

Tris EDTA Purpose

protects DNA by chelating Mg2+

Tris Borate Purpose

carries current in electrophoresis

Tris Buffer Purpose

maintains pH of electrophoresis fluid

Capillary Gel > Slab Gel

more automated, increased sensitivity (up to 1 bp resolution), and can be ran at a higher voltage (run faster as it is more efficient at heat dissipation)

TBE > TAE

TBE has more Tris per liter = greater buffering capacity

recommended for high current or long electrophoresis times and can give sharper resolution

Capillary Gel Electrophoresis

capillary with a negative charge along walls to establish net flow

gel inside acts as sieve and migrates by size and charge

fluorescently labeled, denatured nucleic acid is introduced via electrokinetic injection

high voltage introduced so samples migrate fast

Microfluidic Electrophoresis Purpose

assesses size, integrity, and quantity of DNA

DNA Precipitation Isopropanol < Ethanol

higher DNA concentration to precipitate but salted stays soluble even at cold temp; 2:1 for low concentrations cold and long

TBE < TAE

TBE can inhibit DNA ligase and cause problems if ligation is intended

Lysis Buffer EDTA and Salt Purpose

protect DNA through Na+ positively charged ions, protect negative charged phosphate group on backbone of DNA

Solid Column - Wash 1

low salt, removes proteins and forms cation bridge between negative phosphate backbone of nucleic acid and negative oxygen on silica

Solid Column - Wash 2

high ethanol, removes salts and cation bridge, keeping nucleic acid

Resuspension - Purpose of Dried Pellet

ensures removal of EtOH, that can affect downstream assays and is resuspended in buffer

Resuspension TE vs Water

TE allows samples to be stored better but can interfere with assays

Water is better for lower yields

Nucleotide Deoxyribose and P Location

covalently bound at 1’ C to nitrogen base, G, and a 5’ phosphate group

Nucleotides phosphate on the …. carbon and the hydroxyl group on the … carbon

5’ carbon and the hydroxyl group on the 3’ carbon

rRNA

largest component of cellular RNA (80-90%), important for the structural and functional part of the ribosomes

Secondary Structure of rRNA

important for integrity and function of ribosome, involved in position of ribosome on mRNA and with tRNA during protein synthesis

5’ mRNA Capping Composition

7-methylguanine attached to pre-mRNA by 5’-5’ bond

5’ mRNA Capping Purpose

1. facilitates binding of ribosome

2. increases stability of mRNA,

3. provides resistance to nucleases

4. recognition point for nuclear export and translation

Polyadenylate Pol binds…

AAUAAA on RNA, cleaving 11-30 bases 3’ to site, and polyadenylated end produced by cleavage

Splice Donor Site

at 5’ end of intron includes GU

Splice Acceptor Site

at 3’ end of intron with AG

Splicing RNA Sequence

5’ - exon - splice donor site (GU) - branchpoint (A) - polypyrimidine tract - splice acceptor site (AG) - exon - 3’

tRNA

single stranded polynucleotides, cruciform structure of 4 double stranded stems and 3-4 single-stranded loops

tRNA D loop

recognition site for aminoacyl tRNA synthase

tRNA T loop

recognition site for ribosome

B form diameter

2.37 with two helical grooves for protein binding

rRNA Synthesis Location

in the nucleolus

tRNA Conserved Sequence

most have a G residue at 5’ end and CCA at 3’ end

Replication Overview

1. DNA strands open

2. Daughter strand form

3. Elongation

Topoisomerse

UNWINDS and fixes overwinding of DNA, breaks and reattaches phosphodiester bond

Daughter strand formation step

bases are added via DNAP, adding dNTPs to 3’OH of pre-existing strand forming phosphodiester bond

DNAPIII Requirements

builds on 3’OH, requires RNA primers for nt addition (elongation)

Primase

elongation, synthesizes primers for DNAP nt addition, near fork

DNAPI

elongation, removes RNA primers and fills gap left via 5-3’ exonuclease, leaving bick in backbone of new strand

replaces RNA w DNA nt

DNA ligase

elongation, seals ssDNA nick left after primer removal and fills in via DNAP, sealing phosphodiester backbone

Primers

RNA nt w 3’OH for new DNA nt addition

built by primase and is the starter sequence for DNAPIII

Energy of Replication

nucleotides arrive as dNTPs, providing energy for bonding and breaking of phosphodiester bond

bonded by enzyme: DNA polymerase III (requires Mg2+)

DNAP I / III Exonuclease

edits and proofreads 5’-> 3’

DNAIII on both strands

connected as dimer so replication is continuous

lagging strand loops around it to be in 5→ 3’ position

Pol δ

main lagging strand polymerase with 3’→ 5’ exonuclease, highly processive esp w PCNA

PCNA

processivity factor for Pol δ during replication

Pol ε

main proofreading and leading strand polymerase, attaches to helicase

Pol β

specific repair found in nucleus

Pol γ

replication in mitochondria

DNA Polymerase α

involved in initiation with primase activity

low processivity yet high fidelity

no 3’-5’ exonuclease activity (proofreading),

DNAP α primase activity

synthesizes nt RNA primers and adds dNTPs to the RNA primers

Leading strand synthesis steps

1.DNA Pol α lays down RNA primer and adds DNA

2. DNAP ε binds helicase for DNA unwinding (highly processive)

Lagging strand synthesis

1. RNA primers synthesized by Pol α (10-nt RNA + 10-20-nt DNA)

2. PCNA displaces Pol α

3. δ binds PCNA for DNA synthesis and removes/ fills in the RNA primer downstream of Okazaki fragmen

4. ligase seals the gap

Telomeres

region of repetitive sequences at each end of eukaryotic chromosomes

Telomere Responsibility and Reason

responsible for integrity and stability, due to the mechanism of DNA replication, one strand is left with an incompletely replicated end

Telomerase

adds bases to the ends of chromosomes to replace lost telomeres without DNA template

Transcription

transfer of genetic information from DNA into an RNA via RNAP

antisense strand

template strand, transcribed by DNA

sense strand

coding strand, untranscribed, same sequence as RNA

Transcription Initiation

RNAP binds promoter, determining where transcription starts

Transcription Initiation: Choosing Strand

must choose 3→ 5’ strand for 5’→3’ synthesis

Elongation of Transcription

RNAP untwist DNA helix for DNA nt with RNA nt, adding on the 3’ end of RNA

DNA double helix re-forms

Termination of Transcription

RNAP II transcribes the polyadenylation signal sequence (AAUAAA), and downstream, proteins cut it free from the polymerase = pre-mRNA.

Translation requires:

▪ mRNA

▪ ATP

▪ Enzymes

▪ Transfer RNA (tRNA)

▪ Ribosomes

tRNA Purpose

acts as a molecular interpreter nucleic acid and proteins by carrying amino acids and matching with codons via anticodons

tRNA Charging

specific enzyme for each tRNA< covalent attachment of amino acid to tRNA

Ribosomes

Coordinate the functions of mRNA and tRNA

Translation: Initiation

brings together: mRNA, Met, with its attached tRNA, and ribosomal subunits

requires GTP hlysis

Translation: Initiation Steps

1. mRNA binds to a small ribosomal subunit, then an initiator tRNA binds to the start codon.

2. A large ribosomal subunit binds, creating a functional ribosome.

Translation: Elongation Steps Overview

1. Codon recognition

2. Peptide Bond Formation

3. Translocation

Translation: Elongation - Codon Recognition

the anticodon of an incoming tRNA pairs with the mRNA codon at the A site of the ribosome. – requires GTP for energy

Translation: Elongation - Peptide bond formation

polypeptide leaves the tRNA in the P site and attaches to the amino acid on the tRNA in the A site growing from N → C (formed when carboxyl reacts with amino) via l subunit

Translation: Elongation - Translocation

ribosome moves one codon down the mRNA (5’ to 3’)

tRNA: P site→ E site (leaves the ribosome) → A site (carrying growing polypeptide chain) → P site.

A site is now empty and ready for the next charged tRNA (requires GTP hydrolysis)

repeats until a stop codon is reached

Translation: Termination

elongation continues until: ribosome reaches a stop codon (not charged), completed polypeptide is freed, and ribosome splits into its subunits via release factors and requires E

Translation: Termination- Stop Codons`

UAG UAA UGA

“Wobble”

first two bases form standardly base pairing while third allow looser non-canonical hydrogen bonding

Amino Acid Composition

carboxyl group (-COOH) and an amino group (-NH2)

biochemical properties determined by its side chain (R).

grow from N → C terminus

Amino Acid biochemical properties determined by

by its side chain (R)

Epigenetics

the study of changes in the regulation of gene activity and expression that are independent of gene DNA sequence.

allows rapid adaptation to environmental changes without alteration of the DNA genotype.

Epigenome

the chemical modifications to DNA and histone proteins and the activity of noncoding RNAs that regulate the expression of genes within the genome

Chromatin

DNA is wrapped around complexes of proteins called histones.

Epigenetic mechanisms “open up” or “condense” chromatin altering gene activity without changing gene sequence