Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
74 Cards in this Set
- Front
- Back
|
Basics of Gene Cloning
|
. |
|
|
enzymes coded by bacteria that recognize a specific sequence and cut it |
restriction endonucleases |
|
|
modify DNA at recognition sequence, making it immune to cutting by endonuclease |
DNA methylase |
|
|
Random cleavage, endonuclease and methylase located on the same protein molecule |
Type I and III endonucleases |
|
|
Specific cleavage within recognition site, endonuclease and methylase are separate entities that recognize their own sequence most commonly used enzymes |
Type II endonucleases |
|
|
DNA digestion by restriction endonucleases can produce ____ or ____ ends. |
sticky, blunt (sticky can be 3' or 5' overhang) |
|
|
4 base cutter = ___ base overhang 8 base cutter = ___ base overhang |
2, 3, 4 |
|
|
1) RE digestion of target DNA 2) RE digestion of plasmid vector DNA 3) ligation of target DNA and plasmid vector 4) transform ligation products into E. coli 5) grow on agar plates, with Ab selection |
Steps for plasmid cloning |
|
|
3 options for cloning optimization |
1) use compatible REs 2) directional cloning 3) convert sticky to blunt ends (DNA pol or S1 nuclease) |
|
|
Sources of DNA |
plasmid DNA genomic DNA cDNA (rev. transscribed from mRNA) PCR amplified DNA synthetic DNA |
|
|
Uses of a vector |
carry target gene into host cell maintain target gene in host cell (replicate) express target gene (make protein) |
|
|
types of vectors |
plasmids (bacterial cell) bacteriophage (infect) cosmids/phagemids artificial chromosomes (BAC, YAC, MAC) |
|
|
prototype expression vector for E. coli small ampicillin/x-gal regions |
pUC plasmid |
|
|
contains T7 promoter dependent on T7 polymerase |
pET vectors |
|
|
contain tags to fuse to gene of interest, which can then be easily purified by nickel, antibody, etc. |
Tag vectors |
|
|
clone library |
mix of different clones in the same vector |
|
|
use partial digestion with Sau3A (100bp fragments) and run at multiple time points on gel, then cut, isolate and clone into vector w/ same compatible ends as gene fragment, then select w/ kanamycin on plate |
generation of clone library using gain of function assay |
|
|
ways to clone PCR amplified fragment |
known sequence unknown gene sequence/degenerated gene alteration - introduce restriction sites - generate tag fusions - mutagenesis (site-directed or random) |
|
|
Function of topoisomerases |
cut DNA, allowing winding/unwinding, then religate same DNA ends |
|
|
function of x-gal in vector |
to determine whether gene was inserted color = intact LacZa gene = no insert no color = broken LacZa gene = insert |
|
|
uses phage lambda integrase and excisase pcr fragment flanked by attB/attP sites cloned into plasmid phage and vector mixed together, integrade and excisase added, gene inserted |
Gateway cloning |
|
|
allows determination whether cell has taken up plasmid of interest b/c plasmid confers resistance |
selectable marker |
|
|
Gateway cloning terms: att site |
defined length of DNA = recombination site 4 classes - attB, attP, attR, attL |
|
|
Gateway cloning terms: ccdB gene |
counter selectable gene that allows for negative selection f unwanted by-product plasmids after recombination |
|
|
Gateway cloning terms: donor vector, pDONR |
vector with attP sites flanking counter selectable gene, recombines with gene of interest flanked by attB sites |
|
|
Gateway cloning terms: BP reaction |
recombination b/w attP and attB sites, catalyzed by BP Clonase II |
|
|
Gateway cloning terms: entry clone, pENTR |
vector that contains gene of interest flanked by attL or attR sites |
|
|
Gateway cloning terms: LR reaction |
recombination event between attL and attR sites catalyzed by LR clonase II |
|
|
Gateway cloning terms: destination vector, DEST |
application geared vector with attR sites flanking counterselectable gene that will recombine with one or more entry clones |
|
|
Gateway cloning terms: MultiSite Gateway Technology |
system that allows simultaneous assembly of multiple DNA fragments into a single destination vector |
|
|
hallmarks of the gateway cloning system |
directional cloning maintains reading frame no restriction enzymes no ligation no resequencing b/c few mutations reversible reaction highly efficient and quick |
|
|
TALEN and its applications |
|
|
|
TALE |
"transcription activator-like effector" secreted protein by Xanthomonas encodes TIII effector proteins repeated highly conserved AA sequences, except #12 and 13 KEY FUNCTION = binds specific DNA sequence |
|
|
TALEN |
transcription activator-like effector nuclease |
|
|
TAL effector codes |
#12/13 responsible for base recognition different amino acids recognize GATC on DNA when target is changed, DNA binding not possible until DNA binding portion is changed |
|
|
TAL effectors reaction with DNA |
TAL wraps around DNA #12 = structural support role #13 = specific contact with base left-handed, two-helix bundle |
|
|
TALEN structure and function |
TAL DNA recombined with Fok1 protein when Fok1 is dimerized, cleaves between the 2 TALEN recognition sites |
|
|
Applications of TALEN |
gene knockout (ds cleavage, then host repairs) gene replacement (homologous DNA recom) transcriptional regulation trace chromosomal DNA location |
|
|
How are TALENs delivered? |
plasmid transfection packaged in viral vector mRNA transfection protein delivery (by transfection or TIII SS) |
|
|
Type II RE mediated cloning |
uses BsaI = 6-cutter, cut site is 6bp downstream of recognition sequence digestion product will have sticky ends up to 10bp can be simultaneously cloned into the same vector single tube reaction |
|
|
Golden Gate Reactions |
assembling multiple DNA fragments in an ordered fashion in a single reaction build arrays of 10 repeats each (or less) then join together in a backbone vector by digestion and ligation |
|
|
Gateway mediated TALEN cloning |
has 2 repeating modules that target the G residue pNN (1st step) and pNK (2nd step) either one can be used, equally effective |
|
|
FLASH assembly of TALENs |
select modules, then pre-assemble vectors using BsaI and ligase, then use BpII to bring all of them together |
|
|
Validation of TALENs |
TALEN target is placed on reporter plasmid in yeast system if TALEN protein can target, then reporter plasmid will be destroyed visible when plate transformant and checking for LacZa gene |
|
|
Transposon Insertional Mutagenesis |
.
|
|
|
4 things that all transposons can do |
1) move from plasmid to genome of recipient 2) move from site to site on same genome 3) move from genome to plasmid 4) disrupt genes encoded on insertion sites |
|
|
3 types of Transposition |
1) transposons (dna hopping - cut/copy and paste) 2) Group II mobile intron w/ RNA 3) Retrotransposons (RNA, cDNA, insertion, LINE and SINE in human) |
|
|
3 Types of bacterial transposons |
1) insertion sequence (IS) element 2) composite transposons 3) noncomposite transposons |
|
|
insertion site of a transposon is specifically determined by... |
transposase |
|
|
4 options for delivery of transposons |
transformation transfection conjugation suicide vectors |
|
|
in order to survive, the transposon must... |
incorporate into the chromosome |
|
|
"Tn5" transposase binds inverted repeats, transposon is excised and finds target DNA elsewhere, inserts NO increase in copy number per cell |
"cut and paste" transposition |
|
|
"Tn3" transposon targets DNA, nicks and fills in, then DNA pol fills in other portion of chromosome that is single stranded |
"copy and paste" transposition replicative transposition |
|
|
WT and mutated lambda phage generated, mixed, de- and re-natured = WT, mutated, and WT/mutated. packaged, infected E. coli, selected w/ tet blue = LacZ intact white = defective LacZ blue/white = cut and paste mechanism |
Experimental mechanism to determine which mechanism (cut/paste or replicative) was used to insert Tn10 into chromosome |
|
|
3 general properties of transposons |
target site specificity (by transposase) polar effect on downstream genes (operon - multiple genes) target site immunity (can't insert in same place twice) |
|
|
4 criteria for good transposon library |
high frequency random broad host range selectable marker |
|
|
4 Methods to determine transposon insertion sites |
- direct cloning - Two-step PCR (amplify junction b/w transposon insertion site and target sequence, sequence analysis) - Circulation and PCR (w/ RE cleave non-transposon (ligated/circularized) amplify DNA b/w 2 ends of transposon) - NGS/HTP sequencing (millions of mutants simultaneously) |
|
|
Determination by NGS |
1) mutant library 2) total chromosomal DNA isolation 3) fragmentation 4) anneal artificial primer to 3' end 5) PCR amplify 6) NGS analysis |
|
|
Applications of transposon mutagenesis |
functional gene identification - functional enzymes, regulatory genes, virulence genes, essential genes (good antibiotic target) limiting factors (essential for survival, redundant factors) |
|
|
in vivo version of Tn mutagenesis tag transposon w. specific 20-base signature, generate mutant in S. typhi, inoculate mouse, then analyze original tags and recovered tags. missing mutants = no growth, critical defect (mutated genes encode toxins that kill host immune cells) |
Signature tagged mutagenesis (STM) |
|
|
DNA recombination |
. |
|
|
4 types of recombination |
1) homologous/general 2) nonhomologous/illegitimate 3) site-specific 4) replicative recombination/transposition |
|
|
2 areas of homology join together to form either a chimeric plasmid or integrates into chromosome |
homologous recombination |
|
|
insertion, deletion or inversion can occur seen in attP/attB sites |
site-specific recombination |
|
|
Recombination in E. coli |
RecBCD recognizes DS break, generates 3' tail, pauses at Chi site RecA promotes strand invasion by binding 3' tail RuvAB helicase catalyzes branch migration, removes RecA RuvC endonuclease catalyzes Holliday junction resolution |
|
|
3' ssDNA longer than 5' ssDNA because... |
5' end is degraded faster |
|
|
RecF pathway |
RecQ helicase and RecJ exonuclease bind ssDNA gap, SSB coats gap, then RecF complex (RecF, O, R) bind coated gap and nucleates RecA/displaces SSB, then prepares ssDNA for invasion. |
|
|
Process of _______ 1) exonucleases convert ds break to gap 2) 5' ends degraded 3) 3' end invades homolog in other strand, forms D-loop 4) invading end extended by DNA pol, branch migration, cleavage by RuvC |
Genetic Exchange |
|
|
single step gene knockout |
can occur by homologous recombination (promoted by the RedGem system) - linear DNA introduced by 2 homologous signals at ends |
|
|
Two-step gene knockout |
use SacB selection, where sucrose is converted into a toxic compound and eliminates cells without recombination |
|
|
lambda red recombination system |
3 recombination genes - generate ssDNA gam = inhibits RecBCD, prevents linear DNA degradation bet = acts as RecA exo = exonuclease, generates 3' overhang (only need bet if already ssDNA) |
|
|
Paper 1: one-step inactivation of chromosomal genes in E. coli using PCR products |
simple, efficient easily curable, low copy # disrupts chromosomal genes in which PCR primers provide homology to targeted gene lambda red phage recombinase inducible FLP recombinase encoded by helper plasmids, removes fragments b/w 2 sites |
|
|
|
1) PCR amplify FRT-flanked resistance gene 2) transform strain expressing lambda red recombinase 3) select antibiotic resistant transformants 4) eliminate resistance cassette w/ FLP resistance plasmid |
|
|
Paper 2: |
. |
