DNA Technology

Two main ways to analyze DNA/RNA

1. Agarose Gels (for long strands, runs horizontal)

2. PAGE (for shorter strands, runs vertical, separates based on length/shape (shorter goes down). Two types of PAGE: Native (shape/length based) and Denaturing (adds urea, length based) Shorter and more compact structures run farther. 

Proteins that work on DNA/RNA

Nucleases: cut both DNA and RNA

• DNases: cut only DNA

• RNases cut only RNA

Endonucleases/phosphodiesterase: cut within DNA/RNA

Restriction Endonucleases: cut DNA at specific sequences

Exonucleases: cut nucleotides off the 3’ or 5’ end

Polymerases: synthesize DNA/RNA

Ligases: join two RNA or two DNA strands together

Restriction Endonucleases

cut at Palindrome sequence:

• mirror sequence in opposite strands

produces either blunt or sticky ends

• sticky ends are called that due to the overhangs that occur because of the open area of DNA where another strand of DNA can come in and hydrogen bond/interact with

Sanger DNA Sequencing Method

How we determine the sequence of DNA

ddNTPS are key. 

There’s no 3’-OH nucleophile

requires: primer, template, dCTP, dGTP, dATP, dTTP

Sanger Sequencing Method

1. Synthesize off a primer

2. react with polymerarse, dATP, dCTP, dGTP, aTTP, and a bit of ddTTP

3. Get products.

Polymerase Chain Reaction (PCR)

Makes lot of copies of DNA (doubles the amount of DNA after each round)

Multiple rounds

Process includes:

1. Heat to melt

2. Cool in presence of primers so they anneal

3. Synthesize DNA with Heat Stable Polymerase and dNTPs

Cloning a Gene into an Expression Vector: Simple

1. Cut expression vector with a Restriction Endonucleases

2. Use PCR and RE to engineer a gene to match the gap in the vector

3. Use DNA/Ligase to ligate (connect) gene to vector. Clo

Cloning a Gene into an Expression Vector: Detailed

1. Buy an expression vector (plasmid) that contains all site needed for replication, transcription, and translation in E.coli (ie ori sites, transcription promoter, ribosome binding site) and that contains two restriction endonucleases sites (ie Nde and Bam) that makes sticky ends

2. PCR your gene using the primers that contain the corresponding restriction sites so that you can insert your gene into the gap in the plasmid on the last slide

3. Ligate your gene into the gapped plasmid

Can now insert the plasmid into E. coli and if all goes well, it will replicate, transcribe, and translate your gene into a protein . Then all you have to do is purify the protein. 

Site Directed Mutagenesis

Create a point mutation in the lab

• to do this, use a primer (25 ntds) that contain a forced mismatch in the gene of interest.

• Have a polymerase synthesize the rest of the plasmid (thermocyling like PCR)

• Repeat over and over to get many copies of mutated genes

Primers for Site Directed Mutagenesis are roughly 25 because

1. Specificity: they are unique

2. they’re long enough to overcome the instability of a mismatch.