Recombinant DNA, cloning, genome, editing

molecular editing

technique fundamental to molecular biology and biotechnology that uses living cells (bacteria, yeast) to make many copies of a dna sequence of interest

molecular cloning workflow steps

1) select plasmid

2) target dna osolation

3) create recombinant dna

4) propagate recombinant dna in bacteria or another suitable host

5) screen and select bacteria that express your recombinant dna

6) isolate recombinant dna for further verification and experimentation

1) select genetically engineered plasmid

1) elements of geneticlaly engineeredd plasmids used for molecular cloming

• multiple cloning sites (restriciton sites) - sites with dna sequences recognized by lots of restriction enzymes

• promoter sequence - responsible for regulating rate of transcription; needed or else inserted dna will not be transcribe

• origin of replication - site where plasmid replicates independently

• antibiotic resistance gene (bacterial selection) - only present with circular gene; detects if gene is in plasmid

• selectable marker (mammalian selection)- recognizes their presence in mammalian cells

2) choose which restriction sites u use for cloning

2a Sources of DNA

• tissue biopsy

• cultured cells derived from human tissues

• another expression vector

2b Genomic or cDNA

alternative splicing

introns removed - create isoform of protein which becomes mature mRNA

1 gene codes for different proteins bc extrons are combined in diff combinations

**time consuming

central dogma

genetic material goes from dna—>rna—> protein

2b Target DNA isolation

transcription - dna to pre-mrna —> mRNA by remocing introns —> maturation by adding 5’ end capping and 3’ polyAtail

reverse transcription - isolate mRNA and taek back to double strand, since mature RNA is template cDNA will not have any introns; only protein coding genes are transcribed, so cDNA can be inserted into plasmid

2c Create and amplify cDNA

1) make cDNA

• poly A tail from maturation allows for primes with T nucleotides to bind and thus reverse transcriptase can bind; Ts create oligo primer which is the site for reverse transcriptase which needs a double strand to bind

• reverse transcriptase binds and reads mRNA and sequence of nucleotides adding cmplementary bases and creating cDNA

• restriction sites added to the primer will be complementary to kaiso sequence to attach to end and will have the restriction sites you want to cut out; restriction enzyme BOB will attach to sequence thats the one that should be at the end of the primer, cant have the same sequence inside the gene so not split in half; primers have restriction sites at the end

2) amplify the cDNA

• through PCR repeated 20-40 times

• at the end = billions cDNA kaiso

• vector

• pcr fragment

• vector is cut with the same restriciton enzymes - creates complimentary ends that will align

• add enzymes restriction cretaed by primer to test tube – will cut it and create sticky ends 

mix in test tube needs

• DNA/ RNA  

• Polymerase – also enzyme involved in replciation uses dna template there to isnert nucleotides – willl not bind unless there is primer sequence there like a docking site  

• Primers 

3) create recombinant dna

1) PCR sequence of interest (restriciton sites are added to primer)

2) digest PCR amplicon and cloning vector

3) ligate the insert into vector —> expression construct

• Ligase – another enzyme will ligate and glue the 2 fragments toegther create a single circualr dna plasmid 

Make sure they dont resel on itself

• increase ratio of insert to vector - with somany free floting copies of the isnert chances are reduced that vector resealed on itself

• why you use different restriction enzymes at each end

• bc for a vector only one insert joins the plasmid

4) propagate recombiannt dna in bacteria

1) Take dna from ligation and introduce into bacteria (some such as ecoli are super fast at replicating) 

2) Heat shock will create temporary bacterial – allowing extra cellualr dna to insert and enter into cell  

3) Plasmid dna from ligation will be in transformed bacterium 

• need thousands of copies of this bc we dont know which vector successfuly took in the pcr fragment

What type of marker lets us check if bacteria has taken plasmid

antibiotic resitange gene 

5) screen and select bacteria that express recombinant dna

• antibiotic resistance gene will not be expressed unless the plasmid is circualr

• therefore bacteria when exposed to antibiotic will die if plasmid is linear sicne it wont have the antibiotic resitance gene

• only the plasmid with insert will be circular

• u have to grow it in culture to grow more bacteria from that single colony for each oclony on the plate - and there u add the antibiotic

 

6) isolate recombinant dna for further verifcation 

• Grow them in liquid phase in broth w same antibiotic as plate 

• Will isolate even more bacteria with insert

• Take bacteia from broth open cell membrane and isolate thousands of copies of plasmid that we can now use in other experiments 

• Align sequence we get rfom lab with kaiso sequence to ensure plasmid took it in 

• We take female and male mouse isolate their fertilized eggs and stop it at very early stage

• Before nuclei have fused from male and female 

• take solution with an estimate of the copy number microinject it into pronucleus

• Implant zygote into pseudopregnant mice 

• Make it believe it is alr pregnant – so embryo survives 

• It may reject it – u have to go through multiple rounds 

How has recombinant dna been used

• recombinant monoclonal antibodies - antibodies that are produced using recombinant DNA technology

• subclone coding sequence for antibody u want to create and introduce palsmid to cell line that will produce monoclonal antibody

  (treatment for HER2 breast cancer)

• recombinant vacciens

• human genome project -

How was human genome sequenced

• Gel electrophoresis - Gel and in the lane w unsheared dna (uncut) it would be cut into random fragments (w sound frequecies and enzymes) and cut it into many fragments (shear), we don’t k where it will be cut – creates smear  

• Bc random we cant design primers with restriction sites at the ends  

• So they use blut end cloning 

• Would sequence each clone and align overlapping sequence and assemble genome 

gene editing

alter organism dna sequence by taking advatange cell’s dna repair emchanism

purposes gene editing

• correct mutations

• insert dna sequence

• remove dna sequence (cereating knockouts- loss of protein funciton)

types dna repair processes

• base excision repair - a single incorrect base like A, C, G, or T, would be removed and replaced, single base

• nucleotide excision repair - uses template nucleotide pair as template; more than one base, other components of nucleotide making up a section of DNA; an abnormal bond between bases that could distort the DNA helix, an entire section/ nucleotide needs to be excised to fix

Repairs in dna synthesis during cell division 

• homologous recombination - uses homologue moelcule of dna as template; crossing over bc at some point during ceell division there are 2 copies

• non-homologous end joining - no available template

• 2 broken ends join back together - loss of dna, imapired protein funciton, loss of gene

gene editing uses recombinant enzymes

• aka endonucleases to cleave and alter dna sequences

• endonucleases - porteins and enzyems cut dna (restriction enzymes) 

tools:

• Zinc finger nucleases

• transcription activator-like effector nucleases

• crispr

FOk1

• Non specific restriction enzyme can only function as a dimer 

• Cleaves non discrimitally 

• Not one specific sequence just chops things up unless u tell it where to go 

• A single FOK1 protein wont cut must have 2 of them close to each other -  

• Can be directed to specific sequence by attaching a ZF attache dto it 

zinc finger

• binds 3 nucleotides at a time

• don’t cleave they are more like- flags narrow down part of genome – bind to dna that’s its job – fok 1 is what does the cutting 

• find a ZF that taregts the target sequence and use it

• limiting use bc only binds to 3 at a time

TALEs

• Customizable bc each tale binds to 1 nucleotide 

• Endless number sequences 

• Work same as ZF  

• Their purpose is to bind to dna  

• And guide FOK1  

• Blunt staright down cut both srtands

ZFN and TALENs use FOK1

1) dimerization of FOK1 —> double standed cut —> non homologous repair —> gene knockout

• to replace w new DNA sequence u need 4 ZF/ TALENs

• scientists introduce template so the strand has something to reference - fill nucleotides

CRISPR used for

bacterial immunity

1) Viruses infect bacterial fage – and will isnert and inject dna to proteins cas1 and cas2 

2) Cleaves a part of the fage dna – creates protospacer sequence – itnegrated into bacterial genome – now called a spacer 

3) Fage dna inserts separated by palendromal repeats  

4) cas proteins take a chemical snapshot

5). Crispr array – transcribed single rna strand, matures and combines with cas9 protein

6) complex searches free flaoting genetic material to find any with same sequence as the original sequence

7) compleex scouts, cas9 cleave virus dna protecting bacteria

exploting crispr scientists

• emmanuelle charpentier, jennifer doudna 2020 nobel prize chemistry

crispr-cas9 gene editing

• Synthetic guide rna- SGRNA guides cas9 to region genome we want to edit 

• PAM sequence – is needed there to cleave  

• Cas 9 will go to that region and willl create a doube stranded break 

• That break can be erpair w homologous repair w/ template introduced – we insert additional sequence 

• If it happens we can intiiate knowckout –small deletion 

• If tempalte is provided 

• Cut on tiether side of sequence we want to replace template provided will be used to fill in nucleoties – susbstition 

Sickle Cell Disease Case Study

• Sickel cell blood cell – protection against maleria

• Normal beta globin gene (HbA) 

• Not rounded anymore 

• In babies HbF is expressed not HbA 

• Switch during development 

• HbA predominantes into adult 

• Proteins needed to surpress expression HbF - such as BCL11A - needed for switch 

• Protein surpresses during switch 

• Bc HbS is mutation of HbA 

• Sickle cell disease u don’t have HbA

 

• UK aproved crispr treatment

• Turning off BCL11A – so HbF expression is always on and always expressed 

• Take bone marrow from patient w disease 

• Edit genome. W red blodd cell precurosor  

• Grow cells change/ edit and reintroduce them into patient cell 

• So they will have round hemoglobin 

sickle cell disease pathophysiology

Hba/ Hbs + O2 = round RBC

HbS-O2 = HbS polymerization - sickled RBC clogged blood vessels, impaired blood flow

switch from fetal t adult B globin

HbF (fetal hemoglobin) - pirmary hemoglobin produced in babies before birth

at and after birth HbA predominates (adult)

BCL11A represses transcription of y-globin during fetal adult switch

Born HbS allele

babies w HbS do not manifest

• symptoms appear after compelte HbF - HbS switch

ethics of gene editing

• leverage power of editing to correct disease causing mutations

• - cancer

• HIV

• any other by mutation