10.1 - 10.2 Flashcards
10.1 fragmenting dna
every diploid human cell caries two nealry dinetical sets of 23 chromsomes
each set contains 3 bil base pairs
so avg chromsome is 135 base pairs
this is too much to study
resecrehrs cut the genome into small peices to analzye indivdually
one stategy to frag genome is to use enyzmes that cleave dna at specific base pair seuqneces; altetrive is to frag egenome at rnaodm psooutons by sheairng genomic dna with mehcnaical forces
restriction enzymes cut the genome at sepcific sites
rsearchers use restriction enzymes to cut DNA at specific locations
these cuts allow for manipualtion and charcertization
a restriction enzyme reocgnizes a specific seqeunce of bases anywhere within the gencome and then severs the two phosphidester bodns ta that sequence one in the phopshate backbone of each strand
teh frags generated are reistction frags and teh act of cutting dna is digestion
restriction enzymes can be purfied from bacteral cells
these enzymes digest vrial dna to protetc prokarytic cells from vrial infection
bacteria sheild own gencome by slective adidtion of methyl groups tor estrict recogniziton sites in tehir genomic dna
in teh test tube, retsriction enyzmes from abcteria reocgnize target sequences of four to eight base pairs in dna isolated form any othe rorganism and cut the dna at or near these sites
for the maority of tehse enzymes, the recog site is of 4 - 6 base pairs and tehre palindromic symetry in which base seqeunces of each of the two dna strands are idnetical when read in 5’ to 3’ direction
enzyme can cut at same place on both strings in teh recog seeunce
two ways of cuttiong: right at line of symtrey giving blunt ends or dispalced in opp diretcions from line of sysmetry giving single stranded ends - sticky ends - can base pair with otehr frags cut by same enzyme
disocovery of restriction enzymes
biollist saw that virsues could grow lots on ne strain of bacteria but poorly on anotehr
researchers compared rates of vrial proliferation in terms of plating efefcieny - frction of viral partciles that enetr and replciate isinde host bacterial cells, lysing to release vrial progeny
when a petri dish is coated with continous lawn of bacteiral cells, a virual infetcion forms a cleared spo or plaque where bacteria are eliminated
plating efficeny of virus on ecoli c strain is 1 - 100 virus partciles cause 100 plaques
plating efficneyc on e coli k12 is 0.0001
ability to prevent replication of an infetcing vrius is called retsriction
the vrius that would proliferate - if they grew on anotehr bacth then plating was 1 - this phenomon in which growth on a restricting host modofies a vrius so that suceeding gens grow mroe effcinetly on that same host is called modification
endocnuelase was cutting the dna during restriction - broke phosphidester bonds
called it restriction enzymes
modification enzymes would add methyl groups to dna sequences even on viral stuff
longer the retsriction enzyme recog site, laregr dna frag prodcued
reserachers need to rpoduce dna frags of a partcualr avg length
to make frags of requried size, sicnetsit chosoe approiate restriction enzyme
avg length can be made by asusming two things: that genoem is compsoed equally of all 4 bases and that bases are distributed randomly in sequence
avg distance bewteen reocg sites of any length is then 4 ^n where n is number of bases in recog site
so larger reocg site, the bigger the fragments are in length
1000 bp = 1 kilobase
once u known avg lenth of frags produced by a retsrictioon enzyme u can estimate number of frags produced by treating genome with taht enzyme
is it was 256 base pair size then the human genoem with 3 bill bp exposed to genome gives sizd of 12 mil - (3 bill/ 256 base pairs = 12 mil frags)
enzymes that reocgnzeu larger site prouce fewer frgas of larger avg size
partial digestion with retsriction enyme or mechncial shearing egenrate overlapping genocmic dna frags
sequencing a genome requries that genomic dna frgas overlap with one another
without overlap it would be impsosble to align the base pair sequences of all frags to obtain seeunce of each chromsome
overlap;ping generated when diff copies of genome broken in diff positions
partial resitcion digest do this - they dont cut at all recog sites
altenrtaively, random cutting of DNA can be done by subjecting moelcule to mehcncial stress - niddles at high pressure or ultraosudn enegry
but pulling diff parts in diff drietcion, phsphiedter broken at rnaodm psotions
frags of avg sizes done by changing amoint of mehcncial stress (more, smaller frgas)
end of dna frags of stress are blunt or single strand - not sticky bc random seqeunces - can become sticky or brlunt by using technqiues
gel electrosphersis seps dna frgas accoridng to size
to analyze dna in sample, bilosit use electropherisis, moevemnt of charged moelcules in electrci field
u place solution of dna into wlel at gel matrix, gell in buffer solution, eletric field pushes charged moelcules in opp chrge end (neg dna bc of pohsphate groups move to pos end)
pour agarose into box with comb - allow gel to harden
remove comb, wells are left and place the box into a tank with buffer solution
micripited dna sample into wells
attach electrode to each end for power supply - the opp charge comapred to dna should be on otehr end so neg dna goes to pos end
remove gel from tank, incubate the solution in ethidium bromide which bidns to dna
then expose to uv light - organge bands are dna - estiamte size by comaing to known size markers
variables that affect speed: strength of eletric field applies, compsotion of gel, the charge per unit vol of dna (cahrge dneisty) and physical size of dna
in this its only dna size that afefcts it
long dna moelcule, more volume it occupies
larger volume oocpied, less likley to find a pore in gel to squeeze through and move
larger dna moelcuels move slow comapred to samll
if genome is small, then a small number of band would be seprate eaisly by gell
but eletcrspheris of many diff fragments created like form human dna then tehres a smear centred around ag frag size
pveralpping genomic dna frags geneated by mehcncal sheairng can be sperizted by gel electropehris
frags of size can then be purified by cuting of chunk of gel with dna moelcules - rmeove dna then
diff types of gel used dpending on size of dna frgas being seprated and how diff teh frags are in size
agarose sperates larger frags
polyacrlimde would sperate very minimal difference frags
10.2 cloning dna frags
to dtermine the base pir sequence of every chromsome in human genome, we nee dto sperate each of tehse genomic dna frags from each otehr then make billions of copies of each one
the process that uses lviing cells both to isolate a single dna frag from a compelx mixture and to make exact replcias of teh frag is molecualr cloning
used by human genome prokejct
molecular cloning consists of two basic steps
dna frags are inserted into specilized chrosome like carriers called cloning vectors whihc ensure tarsnport, replciationa dn prufication of idivndual dna inserrts
in seocnd step the combined vector and insert moelcule are introduced into bacteria etc to make copies
bc copies of given frgament are indecal, teh group of rpelicated dna moelcules is knonw as dna clone
dna clonses can be prufied or stored within cells or visues as colelctions of clones known as libraries
ligating inserts to bectors produces reocmb dna
small frags of human dna cant reproduce themslves in a cell
to make rpelciation posisble, its needed to insert each frag into a vector
a vectro must contain two kidns of dna seqeunces: one to provide means of replication for vector and foreign dna inserted and the second to signal the vectors presence to investiagor by concering detcbale proeprt on hsot cell
vector must also have distingusale physical straits like shape by which it can be purified aaya fom the host cell genome
diff vectors are used and each one behaves like a mini chrsomes cpable of accpeting foegin dna inserts and replcating idnepdntly of host clells genome
cuting and ligating of vector and dna frag from two diff orgins created a recomb dna molecule
two characetrcis of stickey ends provide basis for the efficent production of vector/insert recomb: the single stranded overhand allow base pairing and no matetr orgin of dna, teh two stciky ends form same enzyme are compatuble
to make reocmb dan meolcle u can cut the vector with same enzyme as the frag of dna and then u mix the diegsted vector and gencomic dna togetehr in presence of enzyme dna ligase
compelentary sticky ends form base pirs and ligase stablizes it by forming phosphodiester bonds
lab tricks can inc effcieny of utitly of moelcualr clning
certain procedures prevent two or more genomic frags from joing withe ahcotehr ratehr than vector
some minizme chance that vetcor reseals without genomic dna
etc
choice of vectors
available vectors diff form one anotehr in biologcal proeprties, carrying capcity and type of host tehy can infect
simplest vectors are small double stranded dna called plasmids that replciate within bacterial cells
most useful are polylinker - short synthetic dna seqences that conatins diff retriction enzyme sites
each site is ofund one on the polulinker but nowehre else
the polylinekr provides teh felxibility in chocie of enzymes fordigesting the genomic dna to be studies
exposure to one enzyme opens vector at single corepsonding recog site- so insertion of dna with frag cut by same enzyme created linearized by inctact palsmid
circular recomb plasmid
each oaslmid vector had orgin of rep and gene for restance to antibiotic
orgin of rep allows indepdnet replciation in bacteria and gene for resistance allows signal if cell has the plasmdi - seletbale marker
plasmids also fill last requrment of vector - easy prufiictaion
vectors with largest capcity are artifical chrosmoems: recomb dna moelcuels that combine replication and segration el;ements in ways that behave like normal chomsoems when added in cells
bacterial artifical chromsome BAC and yeast artifical chrosmomes YAC allow big dna inserts
host cell take up and amplify recomb dna
vector and human dna cut with same retrsiction enzyme - theyre mixed in presence of dna ligase to create many recomb plasmids eahc with diff frgamnet of the human genome
reseacher than add them into e.coli such that each cell conatins only a single type of recomb dna
transofmratin is when cell takes up foreign dna moelcule, changing gentic charcertics of the cell
scintist add the reocmb dna to e.coli that are snetsive to antibiotic ampiclin
udner certain coditions like cold cacl2 solutn or voltage shock the plasmids enetr 1/1000 cells
this allows permability of bacteiral cell memebrane allowing dna enetcance
to ID the cells with plasmid they are placed in plate conating things including ampicilin - only thsoe with plasmid that had restiance to ampliclin can grow
the orgin of rep anbles it to replciate in the bacteria
eahc vable plasmid containg cell can multiply to produce a dsticnt spot on agar plate: a lcoony of mill cells: collony is a cellular clone
millions of idnetcal palsmid cmoelcules contaiend in colony makeup dna clone
libraries are collections of cloned frags
moving steo by step from dna of any organism to speicfic purified dna frag of tedious
so they build genomic libary: lived collection of clelualr clones that conatin copy of every sequnce in whole genome instered into a vector
scinetist can obtain from libraries, cellualr clones of any region of genome
dtermining which of the clones in librayr contain dna sequnce can be challenging
but once found u can yeild a large anount of the desired genomic frag
if u digested the genome of a isngel human cell with a retsriction enzyme and ligated evry frag to vector with 100% efficney and thne out them into host cells with 100% efficney, reuslting clones owuld rep the entire human genome in a frag form
complete genomic library
number of clones in lirbayr deifnes a genomic equivalent
to find number of cloens that constiyte one genomic equivalent for any library u divide the length of genome by avg size of inserts carried by librarys vector
impsosble to do this irl
eahc step of cloning is far from eficent and dna of single clel doenst supplly suffient raw mateirla for process
reseracher have to harvest genomic dna form mills of cells in organism
a library of four to five genomic evuilanets is prefefred
it would have 4-5 cones of one region of genome and 95% proability that each region is presnet once
