recombinant DNA technology

Question 1

recombinant protein production - why

Answer 1

assay/screen protein for drug activity, SAR from site directed mutagenesis, purify protein for structural studies

Question 2

recombinant protein production - how

Answer 2

choose expression system (bacteria, yeast, human cell, insect cell), design/make expression vector (recombinant DNA/ rRNA), move vector into cells, culture and maintain cells

Question 3

recombinant vectors

Answer 3

vector - nucleic acid molecule used to transfer foreign DNA into host cell, properties - able to have foreign DNA inserted, able to enter host cell, able to replicate in host cell, types of vector - bacterial plasmid, phage, cosmids, viruses and retroviruses, BACs and YACs

Question 4

gene of interest

Answer 4

transcription requires RNApolymerase to synthesis mRNA from DNA template, directed to do this by promoter - region of DNA upstream of gene which can be recognised by RNApol, cells are able to control which genes are transcribed and which are not, most higher organisms’ genes have introns (removed by splicing in post transcriptional modification), usually much longer than exons, having them in vector would add lots of complexity, another post transcriptional modification is addition of 5’ cap and 3’ poly-A tail to mature mRNA, this mature mRNA provides source of DNA we use to express recombinant proteins, we use cDNA to express recombinant proteins, made from mRNA, must use cell type that protein of interest is expressed in to get the mature mRNA

Question 5

making cDNA

Answer 5

mature RNA in cell/tissue easily extracted due to long poly-A tails which can bind poly-T affinity tags, tails useful for binding/directing poly-T primers used to prime reverse transcriptase and produce first strand of cDNA, complementary RNA strand (the mRNA) then partially degraded, leaves hybridised RNA fragments that act as primers to DNApol to make 2nd cDNA strand (like lagging strand of normal replication), these fragments can be placed into vectors and transformed into E coli to produce cDNA (complementary DNA) library

Question 6

gene transfer

Answer 6

mammalian eukaryotic cells (HEK, Cos) usually transfected through lipofection, other methods for more resistant cells (primary cell lines, cultured directly from tissues harder to transfect), electroporation harsher but often necessary for more resistant cells, lipofection reagent cam be complexed with DNA (30 mins) then added to cell culture, lipid encased rDNA can contact cell plasma membrane and be endocytosed releasing the rDNA, if this makes it to nucleus may result in recombinant expression

Question 7

transient or stable recombinant expression

Answer 7

• most transfected DNA that gets to nucleus remains extra-chromosaomal, can be transcribed and translated during life-time of cell (transient transfection), quick to get expression, assay 2-3 days post-transfection, good for getting quick data where limited number of repeats required
• small fraction of DNA can get integrated into cells chromosomes, these can be selected if plasmid used has resistance, DNA survives mitosis and is passed onto daughter cells (stable transfection), slow to generate clonal cell lines due to selection stage, assays can start 4-6 weeks post-transfection, advantage is there only one transfection ,good for HTS

Question 8

bacterial plasmids

Answer 8

contain genes beneficial to bacterial life, usefulness is why they are maintained in bacterial cell despite energy required to produce them, sub-set known as antibiotic resistance genes encode proteins that modify or metabolise antibiotics to inactive form, some genes encode proteins that decrease entry or increase efflux of drugs (fundamental mechanism by which bacterial infections become resistant), some plasmids only exist in low numbers within cell (stringent plasmids) and can be integrated into chromosomal DNA at certain points in cell cycle, others can be maintained at high copy numbers (relaxed plasmids) these are particularly useful in recombinant DNA technology, many engineered relaxed plasmids with variety of specific tailored features now commercially available

Question 9

restriction endonucleases

Answer 9

cleave both strands of double-stranded DNA at specific symmetrical sequences (usually 4-8 bp long) - palindromic, produced by bacteria to defend themselves against invading DNA (from phage), each restriction enzyme is paired with a matching methylase enzyme so the bacteria can methylate their own DNA restriction sites (protect their own DNA from their restriction enzymes), cleavage at opposite sides of axis of symmetry produces sticky ends, cleavage at axis will generate blunt ends, products of restriction digestion will have phosphate groups at 5’ end and -OH group at 3’ end, produce reproducible sets of fragments from a given DNA, need Mg2+ for activity, cleave one phosphodiester bond on each strand, using tow different restriction endonucleases forces the insert to go into the vector in the correct orientation

Question 10

DNA ligase

Answer 10

ATP-dependant enzyme, re-forms phosphodiester bonds between 5’-phosphate and 3’-hydroxyl groups of DNA strand, if strands are already bonded via hydrogen bonds then process quite efficient, can ligate blunt ends together but less efficiently, not sequence specific enzyme so will fix any breaks

Question 11

multiple cloning sites

Answer 11

to make a plasmid a useful as a vector you need a way of inserting foreign DNA into it, multiple cloning sites is a region rich in unique restriction enzyme sites with are not present in the rest of thr plasmid

Question 12

cut and paste sub cloning

Answer 12

one or more restriction endonucleases is used to cut the DNA plasmid in the multiple cloning site, linear DNA with ends compatible to those cut plasmid can be ligated into plasmid using DNA ligase which reformed the covalent bonds, new recombinant plasmid referred to as a construct

Question 13

modification enzymes - DNA ligase

Answer 13

ligase is able to re-ligase the vector, this will happen much more readily then ligation of the insert into the DNA as more. chance of vector ends adhering to each other by hydrogen bonding since they are connected and so always closely located, need a way of avoiding this using modification enzyme called alkaline phosphatase, the phosphorylated 5’ end is critical for ability of DNA ligase to ligate the 5’ end to the 3’ hydroxyl group, however if just one strand is phosphorylated the DNA can still be located so one strand will have a new phosphodiester bond whilst the other remains detached, hydrogen bonding between the two strands maintains DNA structure despite break in DNA chain, this DNA called nicked DNA, can still be transformed and the nick will be repaired once in the cell

Question 14

modification enzymes - alkaline phosphatase

Answer 14

enables dephosphorylation of vector, the two ends have no 5’ phosphates and so cannot re-ligate, however insert sill has 5, phosphates so can still ligate to vector

Question 15

E coli - plasmid factory

Answer 15

standard lab tool bacteria = certain E coli strains, made competent by puncturing cell wall (DMSO or CaCl2), encourage some of the population to take up dan by heat shocking them (ice to 42 degrees to ice) or exposing them to electric field (electroporation), once cells take up new DNA it can replicate within the bacteria cell, referred to as transformed (process of putting DNA into mammalian cells usually referred to as transfection), only some of the cells transformed, plasmid contains antibiotic resistance (often beta lactase - ampicillin resistance), only cells that take up plasmid will survive on agar plate containing ampicillin, each individual bacteria will grow overnight to form colony, colony added to liquid media to grow more bacteria, some of this can be stored in glycerol stock for future use , broth can be used alongside alkaline lysis to extract the plasmid , this should be checked by agarose gel electrophoresis to make sure its the right size and has characteristic restriction sites predicted, can also quantify DNA by checking absorbance at 260 nm (dsDNA at 50 ug/ml results in absorbance of 1), if ok keep glycerol stock san some dan for future use (plasmid factory E coli strains - DH5-alpha or Xl1blue)
(protein production E coli - BL21)

Question 16

culturing E coli

Answer 16

as E coli grows in cultures media becomes more cloudy, follow growth phase by taking out samples and measuring absorbance (600 nm), plotted reveals 4 distinct phases , DNA preps usually carried out on cells in stationary phase after overnight growth (16-18 hours) (amplification of plasmid copies by up to 5 x 10^8), monitoring growth phase more important if planing to produce proteins in E coli, often protein over-expression will impede cell growth so we use an inducible expression system

Question 17

plasmid preps - alkaline lysis

Answer 17

alkaline lysis - SDS, NaOH, and RNAase, neutralise - potassium acetate, aim to separate plasmid DNA, proteins and RNA in cell, care taken to avoid DNA degradation, small preps called mini preps can be carried out with 1.5 mL of E coli culture, larger preps called midi/maxi preps, process the same although mechanics of dealing with larger culture volumes may mean methodology appear a bit different when you do it, dan for use with restriction enzymes, PCR, quickchange, etc can be precipitated in alcohol, transfections of mammalian cell lines requires preliminary stage of plasmid purification, usually using silica-based affinity approaches, before alcohol precipitation

Question 18

agarose gel electrophoresis

Answer 18

DNA is electrophoresed in alkaline buffer - agarose is polysaccharide extracted from sea weed, mixed with alkaline buffer (TAE) melted and poured into appropriate tank with comb that forms the wells, once gel slab set can be placed into electrophoresis tank with more TAE buffer,, dan samples loaded into wells and voltage gradient created across gel, negatively charged DNA migrates to +ve end of gel, small fragments move further/faster, can’t see dan so gel contains ethidium bromide, binds DNA strongly as it intercalates between the stacked base pairs, ethidium bromide fluoresces under UV light and can easily be seen, useful for analytical and preparative purposes

Question 19

linearising circular DNA for agarose gel electrophoresis

Answer 19

sizing circular dan is tricky, linearise it then compare to ladder of pre-prepared standard linear DNA markers, DNA is supercoiled by enzymes in bacteria, when prepped using alkaline lysis usually in a tight ball so runs through gel quick so appears smaller than is, if just one phosphodiester bond breaks the supercoil unravels to form open circular state (nicked DNA), can happen through rough handling (sheer force), the open circular/nicked form runs slowly through gel and appears larger than it is, so restriction enzyme needed to linearise it

Question 20

DNA replication

Answer 20

leading strand - primed once (at the start) the the DNApol simply follows on behind the helicase making new DNA , lagging strand - needs continually priming with primase, leads to fragments of new DNA and requirement for removal of RNA component (original primer) by DNApol 1 (using its 5’ to 3’ exonuclease activity) and its replacement by DNA and the ligation of the Okazaki fragments by DNA ligase to form new phosphodiester bonds to join the nucleic acid backbones

Question 21

DNA hybridisation and primers

Answer 21

if DNA is heated to 95 degrees the hydrogen bonds come apart and the two DNA strands separate, if cooled slowly they can re-find their complimentary sequences and re-form double strand, this is hybridisation (useful property for number of applications - binding a DNA primer on specific location on DNA template, RNA primers essential for DNApol binding as it needs a double strand before it can start making new DNA, in labs DNA primers are used (made in machine - synthetic), DNA primers that are 25-30 bp in length and designed to be complementary to any non-repetitive region of the human genome will statistically only be able to bind to that single region and no other, gives scientists opportunity to direct DNApol to specific sites, 5’ end of new strand will be 5’ end of primer, useful for dideoxy DNA sequencing, PCR and site-directed mutagenesis

Question 22

polymerase chain reaction (PCR)

Answer 22

denaturation/annealing/extension cycle is repeated about 30 times, most proteins are denatured at moderately high temps, DNApol used is special variety cloned from microorganisms from deep vents, stable and can survive 95 degree denaturing stage, primer must be in excess, firstly they get used up as they’re incorporated into new DNA and annealing stage relies on their excess so template strands sent reanneal, 1st round product (derived from original template) has incorrect 3’ ends, DNApol overshoots desired termination point, when 1st round product is the template the 2nd round product terminates in desired place as it falls off this template at its 5’ end, 3rd round product identical to 2nd round product as are all the others, desired PCR product is amplified exponentially and has boundaries defined by 5’ ends of the two primers, because product becomes template the amplification is initially exponential, eventually primers run low and product strands start to anneal to each other rather than primers, h=the strength oof the primer binding is given by their melting temps (Tm) and should be similar for both primers (increases the longer the primer gets and the more GC content), the prime ends need to clamp down so a G or C is best as they have one more hydrogen bond than T or A- primers of 17-25 bp is ok (Tm 55-65)

Question 23

site-directed mutagenesis for drug discovery

Answer 23

drugs bind to target by specific molecular interactions, understanding interactions aids drug discovery, interactions can be identified and quantified by disrupting them and analysing functional consequences, altering protein target achieved by altering coding strand of cDNA

Question 24

transcription

Answer 24

coding strand of DNA is transcribed to give codons on mRNA, template strand of DNA (non-coding strand) runs in opposite direction and is complementary

Question 25

site directed mutagenesis - process

Answer 25

use DNA primer, DNA strands exposed by heating to 95 degrees, cooled in presence of primer (annealing), DNApol uses dNTPs to make complementary chain (extension), 5’ end of new chain incorporates synthetic DNA of the primer as part of new strand, during site-directed mutagenesis a single base change (usually in the middle) is put into the primer, as long as it is long enough (20-30 bp long) it will still anneal to template, primer design stage important as it must be long enough that it bind exactly where required and not in multiple places

Question 26

quickchange mutagenesis

Answer 26

requires two primers, both exactly complementary to each other, bind to template (circular plasmid DNA), make product DNA in opposite directions, denaturation/annealing/extension cycle repeated 20-30, original primers could not amplify more DNA using product as template (could bind product but are pointing in wrong direction so extension can’t occur), all new DNA manufactured directly from template, amplification is linear, the product is plasmid DNA with two nicks where DNApol finished making new strand , nicked DNA is tolerated and repaired in cell, product is unmethylated (template methylated as bacteria methylate their DNA and template extracted form bacteria) so restriction enzyme Dnpl (only recognises methylated DNA) will destroy all un-mutated template, only mutated DNA left,

Question 27

quickchange mutagenesis - PCR machine use

Answer 27

denaturing/annealing/extension cycle repeated 15-20 times, primers can anneal to each other so not in as much excess as in PCR (not a problem as growth isn’t exponential), primer cannot extend from product so product not template like in PCR so more template needed than in PCR, DNA extension continues all way around plasmid until bumps into back (5’end) of primer, product can anneal to form doubly nicked plasmid, template digest by Dnpl, E coli transformed, each colony must e assayed separately - DNA extracted and sequenced

Question 28

Q5 mutagenesis

Answer 28

similar to quickchange but uses reverse PCR, primers not complementary but 5’ ends exactly inline with each other, one primer has the mismatch the other doesn’t, PCR product is linear version on plasmid but with mutation incorporated, DNA made form the template with the non-mismatched primer will not be mutated this will be a minor product, excess primer needed like normal PCR, desires PCR product amplified exponentially and has boundaries defined by 5’ ends of two primers, extension happens as in normal PCR, product is actually linear the ends are blunt and not bonded, template digested with Dnpl, polynucleotide kinase phosphorylates 5’ ends and ligase joins these ends to create circular plasmid, E coli transformed and assessed like normal PCR

Question 29

reverse PCR

Answer 29

can be used with non-mutagenic primers to remove DNA from plasmid by leaving gap between 5’ ends of primers or to add extra DNA at one or both ends of primer, amplifyd product defined by region between 5’ ends of both primers

Question 30

sequencing mutated DNA

Answer 30

primer directed upstream of region to be sequenced, template heated and cooled, primer binds and DNApol adds dNTPs to 3’ end to make new strand, mixture of dNTPs contaminated with ddNTPs, absence of hydroxyl group on 3’ end on ddNTP results in it being chain terminator (last base in DNA chain), since its contaminant will only be incorporated occasionally, idea is generation of all possible DNA fragments that end with chain termination ddNTP, 4 types of ddNTP each has different fluorescent tag, whole sample run on capillary gel, chain terminating fragment detected by type of fluorescence as it passes along gel

Question 31

isolating mutant clone

Answer 31

select clone, transform mixture of different DNA plasmids, results in colonies, each colony contains one type of DNA plasmid/construct, take many separate colonies, grow up 5 mL of each, carry out multiple mini preps, analysed find which contains expected DNA, small prep of correct DNA cast be used to fuel E coli plasmid factory and warehouse to get enough to store

Question 32

stable mammalian cell line - vector

Answer 32

all vectors need to be stored and amplified in bacteria so need antibiotic resistance gene for selection, need origin of replication so bacteria can replicate plasmid and pass to daughter cells, need multiple cloning sites so cDNA can be inserted to right place, depending on expression host vectors require other specific features - promoter must match RNApol of expression host, mammalian expression plasmids primarily used to create mRNA commonly used mammalian terminators include sequence motif AAUAAA which promotes polyadenylation and termination, poly-A tail important for stability of mRNA, protection from degradation and is integral to nuclear export and translation process, mammalian cells can be tricked into replicating plasmids within cells by including viral origin of replication, this is episcopal amplification of plasmids, reduces chances of plasmid being depleted, increasing expression hut still transient transfection (plasmid copied within cell but not passed to daughter cell), for stable expression must be selectable resistance gene

Question 33

sub-cloning stages - selecting restriction enzymes

Answer 33

choose enzymes that - flank your insert not cut within insert, in desired location in recipient plasmid (MCS) and don’t cut anywhere else on plasmid, result in insert being in correct orientation in recipient plasmid, in frame with tags or fusion proteins in recipient plasmid, ideally find two different restriction enzymes

Question 34

sub-cloning stages - gel purification and alkaline phosphatase

Answer 34

DNA fragments gel purified, removes unwanted fragments and restriction enzymes, dephosphorylating the vector prevents re-ligation/ring closure

Question 35

diagnostic restriction enzyme digest

Answer 35

takes advantage of restriction enzymes cleavage DNA at specific restriction sites , often size of plasmid insert and vector backbone are known thus this technique can be used to quickly verify your plasmid, goal of this digest is to cut plasmid into specific size pieces and analyse these fragments by agarose gel electrophoresis, pattern of fragments on gel indicate if plasmid contains expected size insert, choosing correct enzymes allows linearisation of plasmid to determine size of entire construct or extract some or all of an insert from plasmid

Question 36

transfection of plasmid-based rDNA containing cDNA

Answer 36

once expression construct is amplified (containing your cDNA) it can be put into expression host, in drug screening we need meaningful output from analytical pharmacology, cell, cell lines would be mammalian or human-derived (HEK293,CHO,COS),lipofection reagent complexed with plasmid DNA then added to cell culture, lipid incased rDNA can contact cell plasma membrane and be endocytosed releasing rDNA, if it reaches nucleus may result in recombinant expression