RecombinantDNATest2

• 9: RE digest (HindIII and BamHI) of pAMP and pKAN plasmids, then ligation
• 10: Transformation of E. coli with recombinant DNA using classical method (CaCl2 + heat schock)
• 11: Replica plating to ID dual-resistance colonies
• 12: Purification of dual-resistance colonies and determination of which fragments are responsible

10^6

• Easy to work with
• Typically nonpathogenic
• Inexpensive
• Easily transformed

• Bacteria that has been treated (made competent) to take up DNA
• *NOTE - DNA is very hydrophilic and won't normally pass through a cell membrane
• AKA induced transformation

• CaCL2 + heat shock
• Rapidly dividing cells have pores in their membrane
• DNA is suspended in solution of COLD Calcium ion
• DNA forced into cells by incubating cells and DNA together on ice (causing DNA to bind to surface), briefly heat shocking them (42C) (causing a few of the bound DNA molecules to be taken in by the cell), then putting them back on ice

• Lag phase: little growth, activation of genes
• Log phase: rapid growth
• Stationary phase: 
• Death phase: 
• *NOTE- competence occurs late in the log phase of browth

• Check optimal density (OD) of LB + E. coli at 550nm
• Should be between 0.3 and 0.4

• Supercoiled DNA has TE of ~10^6
• Relaxed circular (nicked/covalently closed) has TE of ~10^4
• Linear DNA has TE of ~10^2

• CFU/ug of DNA
• 

• Kanomycin inhibits protein synthesis
• The 1 hour recovery period allow expression of Kan resistance gene product before exposure to the plate

• Satellite colonies are not resistant, but reside in a "zone of resistance" formed by a resistant colony
• This is seen more frequently on LB-AMP, where cells live until wall replication occurs
• on LB-KAN unresistant cells will have already died

• Encyclopedia of DNA Elements
• Follow up to HGP, an effort to catalog all functional elements and regulatory elements in the human genome
• Results: 1.2% of genome codes for exons of protein-coding genes
• 80% is functional (junk DNA not a thing)
• 76% is transcribed to RNA
• 5% is conserved across all mammals (4x higher than 1.2% protein coding region)

• 20687 protein coding genes
• 18400 RNA genes (no protein)
• (8800 small RNAs, 9600 large RNAs (>200nt))
• 11224 pseudogenes

There are ~2.53 protein coding variants per locus (range between 1 and 10)

• sense RNA and antisense RNA worked similarly to suppress genes (no dramatic difference)
• dsRNA was 10x more potent as silencing trigger (RNA interference)
• dsRNA can be "fed" to C. elegans by causing expression in E. coli that they eat

• microRNA: genomically encoded small non-coding RNA that help regulate gene expression
• imp. to early development
• inhibit translation of mRNA bearing complementary target sequence
• Method
• miRNA folds in on itself to form dsRNA
• dicer (RNAse) trims and cuts this dsRNA
• each strand incorporated into RISC
• RISC finds the complementary mRNA and cleaves dsRNA to ssRNA, inhibiting translation

• REQ - recomb plasmid, DNA pol, 1+ primer, all 4 DNA building blocks, 1 tagged ddNTP
• 4 reactions, each contains all 4 dNTPs and 1 ddNTP which is tagged with a radioactive tracer
• *NOTE- dd = dideoxy
• ddNTPs prevent further replication (chain terminator)
• Fragment to be sequenced must be cloned into a plasmid
• Billions of molecules will result in termination at various points in the sequence (A rxn 21, 24, 26; T rxn 23, 29, 33, etc)
• **NOTE - the A reaction is showing that the termination point is a T (the A was added)
• To determination the points of termination, each tube is placed on a HIGH RESOLUTION GEL (this as paper, very long)
• *NOTE - this will allow separation by DNA fragments to a single bp
• Place X-Ray film over gel overnight, silver is displaced by radioactive trackers
• Visualize film on lightbox and read upward for best results

• Automated DNA sequencing
• 4 fluorescent labels (RYBG) rather than a single radioactive tag
• Bases read by a laser/detector rather than by humans
• Only a single lane is required, and as the fragment runs off the end of the gel the laser captures the information!!!
• Data presented in an electropherogram

• Tubes are filled w/ matrix (like a column)
• Mostly completed by robots
• Up to 384 tubes in CE by 2003
• Advantages: no gel pouring, increased # of runs/day, autoloading, robot completion
• *NOTE - no longer used, due to newer tech

• No bacterial clones
• No vectors, no ligase, no transformation
• *NOTE - isolate genomic DNA, fragment the DNA, add dsDNA tag to each end

• fragmented DNA has dsDNA tag added to each end
• suspended beads (single bead in water droplet) have attached primers that are complementary to the DNA tags
• PCR occurs within each droplet (only 15% successful, but others do not interfere)
• Denature DNA -> anneal to bead -> extension of DNA fragment -> denature 1 strand from beat -> repeat)
• break emulsions and deposit beads onto picotiter plate (3.6m wells, only ~1m have bead w/ single frag)
• **NOTE - each company reads the results differently
• company 1 (454 sequencing)
• pyrosequencing
• mix DNA, DNA pol, ATP sulfurylase (enz), APS (subs), luciferase (enz), luciferin (subs), and apyrase (degrades nucleotides)
• 1 rxn creates ATP, other rxn creates light
• integration of nucleotide releases PPi
• PPi + APS -> ATP, ATP + luciferase -> light
• nucelotides added one at a time every 8 minutes until light is seen, then repeated
• *NOTE - apyrase is what degrades the free nucleotides in between each treatment
• Light captured by camera, height = related to # of nucleotide added
• *NOTE - only 400bp per rxn, but 1 million+ rxns

• similar to pyrosequencing
• amplification on "flow cell" (flat) not on a bead
• nucleotides have 4 fluorescent tags
• Flow cell imaged after each addition of 4 nucleotides
• Molecules form "bridges" as they are amplified
• software keeps track of each signal
• *NOTE - 75bp per rxn, 300 million rxns

• very different from other 2 next gen sequencing techniques
• 50bp per rxn, >1 billion simultaneous rxn
• high accuracy (99.94)

• Researchers closely examined the genome of 158 people with brain malformations
• Using sensitive sequencing techniques they covered a depth (how many times each sequence is read) of 300x (more than 5x the normal depth)
• This allowed them to ID mutations that would have otherwise been dismissed as read errors!!!
• Only deep sequencing allowed them to find these mutations! (Sanger would've missed them, and it is still the "golden standard")

• CRISPR - clustered regualrly interspaced short palindromic repeats
• Cas - CRISPR associated proteins
• Cas9 is a nuclease
• First discovered in bacteria, it functions as a defense against foreign DNA (we use Type II)
• It is now a unique tool that allows insertion of DNA askjdnknfa2f

During invasion (viral or plasmid) a portion of the previous invader's DNA is inserted as spacers in the repeat-spacer-repeat-spacer construct

• The CRISPR/Cas system targets foreign DNA with a short complementary ssRNA
• This localizes the Cas9 nuclease and causes a dsbreak, resulting in the silencing of that DNA sequence
• *NOTE - Protospacer Adjacent Motif (PAM) must be present for the binding of the complex

• The genes for crRNA and tracrRNA can be JOINED creating a single GuideRNA which is functional!!!! (simply add a loop to bind them together)
• This works in eukaryotic cells - and the ds break can be repaired (or inserted :) )
• Only the target sequence need be changed to target specific genes!

Genes can be "knocked out" to determine function

• Have AmpR and KanR
• Have origin of replication (2 is ok)
• Hind to Hind, Bam to Bam
• Must be circular
• Limit 4 peices (large size prevents entry to E. coli)
• Same pieces cannot be "next door neighbors"

• Super plasmid (all 4)
• simple recombinant (only 2 required)

• Supercoiled (will travel furthest, only one to compare)
• nicked
• multimer

• Complementary strands will occur both next to and across from a given strand
• this causes a loop to form, and replication is unlikely

• Helps to verify that we have DNA, not protein
• Concentration [ng/uL]
• 260/280 ~ 1.8 for DNA
• 260/230 ~ 2-2.2 for nucleic acids

To remove remaining RE and RE buffer that would interfere with the next digest.