Lecture 22: Recombinant DNA Tech

Recombinant dna tech

Topic 22

  1. these techs allow us to engineer living organism to have desirable properties or abilities
  2. techs moved us into era synthetic biology


FoxP2 Gene → Guided Example

  1. patients struggle with complete sentences and tongue movements
  2. gene located on chromosome 7
  3. want to clone gene to:
  4. determine sequence
  5. purify protein and investigate properties
  6. clone → copy of original
  7. E.coli commonly used for cloning

Expressing FoxP2 in E.Coli

  1. determine which tissues express FoxP2
  2. isolate mRNA
  3. synthesize cDNA
  4. clone it into expression construct
  5. purify FoxP2 from E.coli
  6. two common methods check gene expression
  7. Quantitative Real Time PCR < short qRT-PC
  8. Northern Blotting


Blotting → separating DNA, RNA, proteins by size then transfer onto a membrane

  1. probing for species of interest and visualizing

Southern:

Looking For: DNA

Using: DNA

Northern:

Looking For: RNA

Using: DNA

Western:

Looking For: protein

Using: antibodies


Northern Blot

  1. Isolate RNA
  2. have ways of isolating → not important
  3. Separate RNA by gel electrophoresis
  4. RNA → contains all mRNA from cell
  5. mRNA's are different sizes
  6. gel electrophoresis → separates by size
  7. principle same SDS-PAGE (proteins) except RNA already (-) charged (cause phosphates backbone)
  8. another difference → don't need detergent to denature can just use warm temperature
  9. can use polyacrylamide or gels of agarose (polysaccarides) to separate RNA/DNA
  10. To Run Agarose Gel:
  11. need to put RNA samples in individual wells at one end of the gel
  12. cover gel with buffer → then apply electric current
  13. (-) charged RNA moves
  14. away from (-)
  15. towards (+) electrode
  16. smaller pieces RNA move faster than larger
  17. after some time fragments will be separated
  18. can't see with naked eye
  19. have to add dye (Ex. Ethidium Bromide)
  20. when shine UV at dye bound RNA/DNA glows
  21. Transfer RNA to membrane
  22. for northern blotting don't add dye
  23. instead transfer RNA from gel → membrane
  24. take gel with separated RNA and lay nitrocellulose or nylon membrane flat on top
  25. put gel and membrane on sponge sitting in salt solute
  26. stack some dry paper towels on top
  27. salt solution moves up paper towels by capillary action
  28. RNA moves along with it out of gel → membrane
  29. eventually, all RNA will be transferred to the membrane
  30. Probe Membrane for FoxP2
  31. strands denature → when heated
  32. stands anneal (renature) → when cooled
  33. after RNA on membrane no longer denature and can form secondary structures (hybrids DNA)
  34. first, denature any RNA secondary structures
  35. anneal DNA probe complementary to the sequence of interest to RNA
  36. forming DNA/RNA hyrbid
  37. to form hybrid, synthesize an oligonucleotide probe that's complementary to part mRNA your looking for
  38. assume sequence known (human genome sequenced)
  39. add radioactive phos group to 5' end probe
  40. incubate membrane (with RNA on it) with probe under condition allow specific annealing probe to complementary strands but not others
  41. wash away unbound probe and detect radioactivity on membrane
  42. if you see band in lane, the RNA of the desired gene is present

Once you have identified tissue type mRNA is expressed in

  1. use tissue as source make many copies of your mRNA


Microarray

  1. another way looking specific mRNAs
  2. allows look mRNAs different genes at the same time
  3. on 2D surface attach oligonucs complement to different mRNAs
  4. each oligonuc different spot on surface
  5. tag RNA prep with fluorescent dye
  6. RNA fluorescently labelled
  7. mRNA labelled not probe
  8. incubate surface with your RNA prep
  9. if certain mRNA present will bing oligenucs in spot that corresponds specific mRNA
  10. each bright spot → specific mRNA
  11. each dark spot → mRNA not present


cDNA - Complementary DNA to RNA Strand

  1. DNA copy mRNA, needed for cloning
  2. allows amplification sequence
  3. start with total mRNA prep
  4. synthesize cDNA using reverse transcriptase and poly (T) primer
  5. reverse transcriptase found RNA viruses (covid)
  6. synthesizes DNA using RNA, DNA poly can't
  7. use poly T oligonucleotide as a primer which is complementary to poly-A tail mRNAs
  8. end up with DNA and RNA hybrid
  9. to get double stranded DNA → add RNase enzyme to degrade original mRNA
  10. same tube you included DNA poly and deoxynuc triphosphate
  11. polymerase use partially digested to mRNA as primer to synthesize second strand DNA

  1. first mRNA strand add poly-T primer
  2. can anneal 3' end mRNA
  3. including reverse transcriptase in mix results in synthesis of DNA strand that comp to original mRNA strand
  4. reverse transcribe all mRNAs
  5. not gene specific

Gene Specific Part

  1. we add 3 enzymes
  2. RNase H specifically degrades RNA bound comp DNA strand
  3. will degrade RNA random spots
  4. partial segments RNA bound DNA
  5. RNA fragments act as primers for DNA polymerase
  6. DNA poly → synthesizes new strand
  7. RNA ligase join together all nicks as all RNA degrades to → DNA
  8. end up with double stranded cDNA
  9. representing all mRNAs in starting tissue


PCR → Polymerase Chain Reaction

  1. amplifies → many copies made from very small amounts of starting DNA
  2. little as single copy target DNA
  3. How it works:
  4. DNA presented (the cDNA or genomic) with target region to be amplified in darker colours
  5. dark region → entire coding sequence of gene (FoxP2)
  6. rxn mix → has two primers
  7. they are comp to template strands at either end of region to be amplified
  8. must include DNA polymerase to synthesize new DNA
  9. polymerase → must be thermostable
  10. include deoxynucleotide triphosphates
  11. as substrates for DNA polymerase
  12. appropriate buffer to provide conditions under which polymerase will be active


To Perform PCR change Temp → 3 Stages:

  1. and repeat many many cycles
  2. raise temp very high around 95 °C (Separate 95°C)
  3. causes all DNA in solution to denature or to separate into two strands
  4. need poly to withstand this
  5. cool DNA moderate temp (Anneal 50-60 °C)
  6. allows primers to anneal specifically to complement
  7. 3' ends both primers toward region to be amplified
  8. raise temperature optimal for activity of polymerase (Extend 72 °C)
  9. around 72 °C
  10. add nucs 3' end of both primers resulting in duplication of DNA in region to be amplified


  1. programable thermocyclers invented automatic
  2. DNA strands that were synthesized in the first cycle become templates for new DNA synthesis in the second cycle
  3. if everything perfect → double the number of copies of each cycle
  4. starting cycle 3
  5. precise target region grow in abundance until soon major product
  6. after 30 cycles will have 230 copies target DNA
  7. if you analyze PCR product using gel electrophoresis
  8. see single band representing cDNA you want
  9. can cut band out of gel and extract DNA from it
  10. gives you fairly pure sample cDNA
  11. next step cloning

Application of PCR

  1. Quantitative PCR (qPCR)
  2. molecule that becomes fluorescent presence double stranded DNA
  3. fluorescent gets brighter as more amplification
  4. can use to quantify amount specific mRNA
  5. DNA Fingerprinting
  6. humans repetitive regions in DNA that contain repeats short sequences
  7. number of short depends on the person
  8. more you check, less likely unrelated
  9. used crime scenes and paternity tests
  10. Site Directed Mutagenesis
  11. used introduce desired changes in DNA sequence
  12. design primer not completely comp to template sequence
  13. contains one or more mismatches
  14. ensure 3' end primer anneal template and used by DNA polymerase
  15. as PCR progresses altered version dominant product


Plasmids

  1. relatively small pieces DNA found in microorganisms
  2. not part of organisms genome
  3. usually circular, double stranded, less than 1000 base pairs in size
  4. multiple copies of single plasmid present in bacteria
  5. convenient vehicles/vectors → gene can be inserted

Plasmid Features

  1. must contain replication origin
  2. very helpful have selectable marker
  3. to help distinguish
  4. often gene that confers resistance to an antibiotic
  5. if cells grown media containing antibiotic cells that don't have die, and ones that do have gene survive
  6. many have multiple cloning regions
  7. for section DNA contains recognition sequences for many restriction endonucleases (restriction enzymes)

Restriction Endonucleases

  1. help with ability to manipulate DNA
  2. usually recognize specific motifs in double stranded DNA
  3. usually between 4-8 bases long
  4. common one can generate sticky ends which
  5. have 5' or 3' overhang or blunt ends

Creating Recombinant DNA

  1. multiple cloning site has many restriction endonucs recognition sites
  2. allow cut open circular DNA and past exogenous/foreign DNA (compatible ends)

  1. cut plasmid and DNA gene with restriction enzymes
  2. using overhangs/sticky ends
  3. incubate fragments together to allow sticky ends to anneal
  4. adding DNA ligase → backbone repaired
  5. needs 5' phos present in order to join strands
  6. result intact circular DNA that contains desired insert

Example: BamHI from Bacillus Mega HI site on Plasmid to insert cDNA Gene

  1. add BamHI cut site onto end of cDNA by ligating on short pieces DNA that contain that DNA → linker DNA
  2. OR design PCR primer containing BamHI recognition Site
  3. gives rise sticky ends compatible
  4. purify fragments away from restriction enzymes
  5. mix fragments
  6. add DNA ligases
  7. some frequent insert ligated into plasmid giving rise to desired recombinant plasmid
  8. plasmid introduced E.Coli for protein production


Transformation

  1. process which plasmid introduced to bacteria
  2. process bacteria takes up exogenous DNA

Two Main Ways Transform Bacteria:

  1. Electroporation
  2. mix plasmid and bacteria under certain conditions
  3. then expose cells to electric current
  4. causes some cells uptake plasmid
  5. like shooting little holes into bacteria cell wall
  6. Treating Bacteria with chemicals
  7. often divalent cations (ex. calcium) make them;
  8. competent → cells capable of taking up foreign DNA
  9. mix cells and plasmid together
  10. quickly warming cells up in "heat shock" induces some cells to take up plasmid

*for both methods: must be circular DNA*

  1. to be stable in bacteria
  2. linear doesn't survive in bacteria


Selection

  1. incubate cells on an agar plate containing antibiotics
  2. that corresponds selectable marker on plasmids
  3. cell contains plasmid → resistant antibiotic
  4. will grow on plate
  5. no guaranatee that plasmid contains insert wanted no guarantee that plasmid contains insert wanted
  6. maybe ends of plasmid cut were ligated back together without insert
  7. would be resistant but not have cDNA wanted
  8. must test colonies find one contains recombinant plasmid
  9. each method grow up culture of colony and isolate plasmid DNA from cells

Restriction Mapping (check insert technique)

  1. cute plasmid with particular restriction enzymes then run products on gel and check sizes
  2. know sequence plasmid → can predict fragment size
  3. can tell insert about right size

Example: Two EcoRI sites (500 bases apart)

  1. add 1000 bases insert between
  2. if you cut recombinant plasmid → expect 1500 bases
  3. visualize by separating pieces by size (like northern blot)
  4. have to stain gel to see DNA
  5. use dye called ethidium bromide
  6. binds DNA (the dye) and glows under UV light

Expect to See: 500 (Eco RI Sites), 1000 (insert) and 2500 (set of plasmid - total was 4000) base blot

Clones Must be Verified

  1. PCR → can be used to check for correct insertion
  2. use primers that flank predicted insert
  3. if you see PCR product appropriate size that evidence your plasmid contains insert

Third Way Check Insert Presence Clones

  1. denature plasmid and incubate it with oligonuc probe specific to insert sequence
  2. can be done by southern blotting
  3. if probe binds to plasmid →suggests insert is present

*all three methods are no guarantee*


Sanger DNA Sequencing (dideoxy)

  1. chain termination sequencing
  2. best way to test and verify plasmid is what you want it to be
  3. set up DNA synthesis rxn with primer that anneals to DNA thats to be sequences
  4. and DNA poly and deoxynuc triphophates
  5. include dideoxy NTPS → "chain terminators"
  6. lack hydroxyl at both 2' and 5' carbons sugar
  7. Effect DNA replication
  8. dideoxy NTP incorporated into growing chain
  9. DNA synthesis stops at that point
  10. no hydroxyl to add to
  11. include bit of all four dideoxy NTP
  12. DNA replication can stop anywhere
  13. each base different fluorescent label

*Method useful relatively in small number of samples to analyze over limited length sequence*


ddATP RXN

RXN Mix

  1. template, one primer (anneals to template), DNA polymerase and deoxy NTPs (so synthesis occurs)
  2. include 0.2% dedeoxy ATP, compared regular deoxy ATP
  3. dideoxy ATP modified by fluorescent label
  4. DNA poly binds to template and starts adding nucs to the primer
  5. when comes to T on template incorporates A
  6. 0.27 chance dideoxy incorporated
  7. DNA synthesis stops if that happens
  8. millions of DNA templates
  9. all newly synthesized strands NOT uniform
  10. all fluorescently labelled at the end (A)
  11. end up with series of newly synthesized

DNA fragments

  1. each stopping at different points in sequence and each fluorescently tagged according to last base added (differs)


Analysis

  1. denature strands and separate by capillary gel electrophoresis → in tiny tube
  2. smaller and quicker
  3. monitor bottom capillary for fluorescence
  4. can read complement DNA strand want sequence
  5. get output with peaks
  6. each peak represents signal from strand
  7. computer converts peak → sequence
  8. confirms whether cDNA in plasmid or not and sequence given to analyze


Purifying Protein from E.Coli

  1. Create Expression Plasmid
  2. Transform cells with plasmid
  3. Grow cell culture → Inducing OverExpression
  4. grow cells containing liquid culture and induce high level expression gen
  5. Lyse Cells
  6. when having a lot of cells, lyse them (break)
  7. use physical methods to disrupt cell wall/membrane
  8. Remove Cellular Debris
  9. use centrifugation to remove remanents
  10. Chromatography
  11. size, charge properties, affinity matrix
  12. left with complicated mixture
  13. to separate protein from other molecules use chromatography
  14. Check Purity
  15. of protein preparation

Anatomy Protein Expression Vector

  1. Vector → something used to move some DNA interest into desired location
  2. means introducing cargo DNA to system
  3. plasmid must have origin replication
  4. some origins have many copies. some only few copies
  5. Promoter
  6. bacterial RNA poly can recognize
  7. contain regulatory sequences allow transcripts turned on
  8. mRNA has ribosome binding site
  9. so mRNA can be translate
  10. affinity tags
  11. simplify purification
  12. put 3' or 5' and fount at N/C terminus
  13. tags are usually couple residues long
  14. His tags binds nickle ions - immobilize in chromatography column
  15. Tag Removal Sequence
  16. sometimes don't have to remove - so small
  17. usually sequence includes protease coding
  18. Terminator
  19. knows poly when stop
  20. Selectable Marker
  21. to identify cells with plasmid

Affinity Purification

  1. equilibrate material binds affinity tag to ensure everything same buffer
  2. polyHis tag → Nickle affinity chromatography
  3. load sample onto column
  4. only protein interest bound column
  5. elute protein interest by adding competitive (amidsole) binder
  6. protein now half pure

SDS-Page Analysis Example Purification

  1. Cell Extract
  2. some cells over express and broken open
  3. Flow through Fractions from Affinity Column
  4. Purified Protein
  5. analyze structure/function to see how it works


Transgenic Organisms

  1. organisms whose genomes have been permanently altered by genetic engineering
  2. plasmid not transgenic because not part of genome
  3. vital studying genes and proteins in context of whole organism
  4. Three main types changes to gene:
  5. gene replacement
  6. gene knockout
  7. gene addition (higher expression)
  8. observing changes → valuable information for function


CRISPR - Cas9 → Clustered Regulatory Interspaced Short Palindromic Repeats - Endonuclease

  1. Cas 9 → creates double stranded breaks
  2. cuts specific sites DNA by guide RNA (binds enzyme)
  3. without guide, no cuts
  4. researchers cut specific → provide right guide
  5. Guide → 30 nucs long, 20 nucs specify cutting
  6. very unlikely targeted 20 nucs present more than once in genome cause 420 random bases

After Double Stand Breaks:

  1. double strand machinery tries fix break
  2. non-homologous:
  3. likely bases lost at repair site
  4. if near start gene, good chance gene inactivated to create gene knock-out
  5. homologous:
  6. if replacement DNA provided chance organism will copy that during repair
  7. altered version gene created
  8. alternatively, entire gene could be added site repair

Mice with Altered FOXP2 genes

  1. Homozygous Knockout
  2. develop delay, motor abnormal → premature die 3-9 weeks
  3. fewer vocalizations upon separation from mother
  4. Heterozygous Knockout
  5. fewer vocalizations upon separation from mother
  6. different male courtship songs



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