Molecular Methods: DNA

polymerase chain reaction (PCR)

amplify DNA sequences

1. dsDNA template

2. flanking, inward-facing, oligonucleotide primers

3. thermostable DNA polymerase

4. dNTPs

needed for PCR

thermostable DNA polymerase

can withstand high temps without breaking down or losing function, can survive heating steps in PCR that melt double strands without denaturing

1. denature at 100 C

2. anneal at 55 C

3. polymerize at 72 C

4. repeat

PCR amplification steps

agarose, ethidium bromide

use these for DNA electrophoresis (dif from protein) - ___ instead of polyacrymalide, __ instead of coomassie

terminator ddNTPS

added to growing nucleic acid but cannot be extended bc they lac a 3’ -OH

Sanger sequencing

chain-termination method where ddNTPS prevent letters being added to end

1. window into protein expression

2. universal reagents, no need for specialized antibodies

3. high throughput methods

why measure RNA?

1. RNA unstable and rapidly degraded

2. tools for DNA sequencing/manipulation are better

challenges for RNA analysis

complementary DNA (cDNA) synthesis

single-stranded DNA made from RNA template, more stable than RNA and can be copied and sequenced easily

reverse transcriptase

viral enzyme that has terminal transferase activity adding 2-3 cytosines

terminal transferase activity

enzyme that can add random nucleotides to very end of DNA strand without needing template to guide it

first strand synthesis

copying RNA template into cDNA

second strand synthesis

replacing original RNA strand with cDNA to create stable, double-stranded cDNA molecule (dsDNA)

qPCR/real time PCR

allows scientists to measure exact amount of DNA/RNA in a sample in real time as rxn occurs, based on fluorescence resonance energy transfer (FRET)

RNA sequencing (RNA-seq)

analyzes entire collection of RNA molecules in a cell, tissue, or organism by running sample through NGS machine that sequences millions of fragments simultaneously

single cell RNA sequencing variation

allows scientists to examine gene expression profile of individual cells

in situ hybridization

used to detect and locate specific DNA or RNA sequences directly inside cells where gene is being actively expressed by labeling DNA/RNA piece (the probe) with fluorophore and observing under microscope

1. origin of replication

2. antibiotic resistance gene

3. multiple cloning site

all plasmids must have:

DNA construct

engineered genetic material grown in microorganisms (preferrably E. coli), tricks bacteria to depend on DNA construct for survival

restriction enzyme

sequence-specific DNA endonucleases that recognize palindromic sequences (targets) and cut DNA