MMG115 Midterm 1

recombinant DNA technology

involves using enzymes and lab techniques to manipulate DNA and propagate the recombinant DNA segments

genetic engineering

the process of using recombinant DNA to alter the DNA makeup of an organism

e.g. for production of recombinant human insulin or growth hormone in bacterial cells

steps of genetic engineering

1. Identifying the gene(s) or regulatory regions of genes that determine a particular trait

2. linking the gene of interest to a suitable vector

3. introducing the recombinant DNA into a different host organism

4. propagating the engineered organism that carries the recombinant gene (i.e. the new trait)

what can genetic engineering used for?

combining or deleting DNA from different species or to create genes with new functions

recombinant insulin production

1. DNA from a human pancreas cell is isolated

2. a plasmid DNA cut with restriction enzymes is isolated

3. a ligase is used to ligate the two pieces of DNA together to form a recombinant DNA

4. the recombinant DNA is then introduced into a bacterial cell, i.e. E. coli

5. the bacterium with the recombinant DNA is placed in a fermentation tank and is multiplying and producing insulin in fermentation tank

6. the human insulin is then extracted and purified, allowing it to be produced in large quantities

DNA

nucleotides linked together w phosphodiester bonds

how are complementary strands of DNA held together

• hydrogen bonding

  • GC bonds are more stronger than AT bonds because GC bonds are 3 hydrogen bonds

• base stacking (hydrophobic interactions)

reverse transcriptase

an enzyme that can create a DNA strand from an RNA template, retroviruses have reverse transcriptase. eukaryotes also have this enzyme in the form of telomerase, that uses RNA to make bits of DNA

• rna dependent dna polymerase

• uses single stranded rna or dna templates

• polymerizes 5’ to 3’

• requires rna or dna primer

• no exonuclease activity

  • since it has no exonuclease activity, it is prone to error

  • most likely did not evolve exonuclease activity b/c not involved in super long pieces of DNA

semi-conservative DNA replication

each strand can act as a template --> one parental DNA produces two daughter DNA each with an old and new strand

direction of DNA synthesis

The new strand is always synthesized 5’ to 3’

primase

synthesizes a primer which provides a starting point of RNA for DNA polymerase to begin synthesis

DNA polymerase

syntheiszes new strands by adding complementary nucleotides

helicase

opens the double helix by breaking H bonds

SSB proteins

bind to ssDNA at the replication fork to prevent the DNA from reannealing

topoisomerase

relaxes the DNA from it's supercoiled nature

DNA ligase

catalyzes end joining by forming phosphodiester bonds

DNA pol III

the workhorse that carries out replication of the genome

- polymerase 5' to 3'

- requires RNA or DNA primer with a 3'OH group

- can be used to fill in gaps, make new DNA and convert overhangs to blunt-ended DNA

- at least 10 different subunits

DNA pol I

removes the RNA primer and fills gaps between Okazaki Fragments

- 5' to 3' polymerase activity: requires primer with free 3' OH, Mg²⁺, and dNTP

- 5' to 3' exonuclease activity: requires dsDNA and Mg²⁺, only active when the 5’ to 3’ polymerase activity is on

• can be separated from polymerase activity by protease cleavage or by genetic engineering to generate the Klenow fragment

- 3' to 5' exonuclease activity: proofreading

- made up of a single polypeptide chain, can easily produce the active enzyme in large quantities, therefore is more used in laboratories

Klenow Fragment

a fragment on DNA Polymerase I that consists of the 5’ - 3’ polymerase and the 3’ - 5’ exonuclease. it sits towards the C terminus, and can be genetically engineered. the 5’ - 3’ exonuclease is not included in this fragment

The Nick Translation Reaction

5’ to 3’ exonuclease activity of E. coli DNAP 1 works with the 5’ to 3’ polymerase activity. DNAP 1 can bind to where the nick in the DNA strand is, and continue chewing off and polymerizing the rest of the strand toward the 3’ end.

This reaction can be used to label a DNA fragment with fluorescent or radioactive labels

common features of DNA polymerases

• they use single stranded templates

• they require RNA or DNA primer with a 3’ OH group to start polymerization

• they polymerize the new strand in the direction of 5’ to 3’

T4 DNA polymerase

- DNA dependent polymerase

- 5' to 3' polymerase activity

- strong 3' to 5' exonuclease activity —> strong proofreading activity

- NO 5’ to 3’ exonuclease activity

- requires a primer

primer annealing

process of primer finding its correct target sequence

Tm

melting temperature where half the DNA is in double stranded form and half in single stranded

nucleic acid hybridization

two complementary single-stranded nucleic acid molecules (DNA or RNA) bind together through hydrogen bonding to form a double-stranded duplex

helix nucleation

a rare random collision juxtaposes complementary nucleotide sequences on two ss-nucleic acids allowing the formation of a short stretch of base-paired nucleic acids, this is part of nucleic acid hybridization

rapid zippering

leads to successful annealing with base pairing between the rest of the complementary sequence after helix nucleation

factors that influence hybridization

temperature, salt concentration, detergent concentration, and organic solvent

factors that constitute the higher stringency of hybridization

• higher temperature

• higher detergent concentration

• higher organic solvent concentration

• lower salt concentration

at less stringent hybridization conditions, even when two nucleic acid strands contain some base pair mismatches, they can still form stable hybrids

DNA sequence detection using a Probe

when a dsDNA strand is heated, it will denature. if we add a dna probe that is complementary to the sequence we are looking for, it will bind the target dna strand and form a double stranded hybrid molecule

components of PCR

DNA pol (Taq), two primers that bracket target DNA sequence, dNTPs, buffer, DNA template

steps for PCR

1. denaturation (94-98): strands separate

2. annealing (50-65, around 5 to 10 degrees C below Tm): hybridization of primers

3. extension (68-72): synthesis of new strands

First Established PCR machine

• used klenow fragment

• unstable with heat

• three water baths to maintain the different temperatures

• cycling involved manual transfer of the samples from one water bath to another at specific times

• new enzyme had to be added manually at each step

• maximum length it could reproduce was 400 bp

Taq DNA Polymerase

• first isolated from thermophilic bacteria

• active at higher temperatures

• optimum temperature for activity is 75-80 degrees C

  • has a half life of greater than 2 hours at 92.5 degrees celsius, 40 minutes at 95 celsius, 9 minutes at 97.5 celsius

• has 5’ to 3’ polymerase activity

• has 5’ to 3’ exonuclease activity

• no 3’ to 5’ exonuclease activity, therefore has a relatively high error rate

5’ to 3’ exonuclease vs 3’ to 5’ exonuclease

5’ to 3’ exonuclease recognizes a free 5’ end, then cuts adjacent phosphodiester bond

3’ to 5’ exonuclease recognizes a free 3’ end, then cuts adjacent phosphodiester bond

what proteins are needed in replication but not in PCR

primase, helicase, SSB, topoisomerase, ligase

conventional PCR

- aka endpoint PCR

- endpoint measurement done by gel electrophoresis and staining after PCR is complete

• used if you just want to make lots of product without trying to measure starting material

real time PCR

- aka QPCR

- continuous measurement allows you to determine when the reaction enters the exponential amplification stage and the plateau stage

- analysis of the exponential/log phase allows quantification of starting amount of given DNA sequence

• can’t use plateau stage to determine because identical samples can produce different quantities of reaction products by the plateau phase of PCR. meanwhile the threshold cycle values remain consistent

• if one test tube takes you less cycles to read the threshold, that means there is more sample in the that test tube compared to your other test tubes

methods of detection of PCR products in quantitative PCR

1. non-specific fluorescent dyes that bind any dsDNA: SYBR green-based PCR

2. sequence specific DNA probes with fluorescence, permits detection only after hybridization of the probe with its complementary sequence: TaqMan probe-based PCR

threshold cycle

- CT is the number of cycles for any one reaction to reach the threshold fluorescence

- The value of CT is inversely proportional to the log of the initial target DNA concentration

- the threshold is set in the exponential phase of the amplification plot

- the imager used is limited by sensitivity, so after a couple of cycles, the imager will be able to pick up the fluorescence

SYBR green

• binds to the minor groove of DNA and fluorescence is amplified by 1000fold after binding to dsDNA

• fluoresces green under blue light

• we use an imager to help measure product

• the number of fluorescence is proportional to the number of dsDNA

  • amount of SYBR green fluorescence is measured at the end of the extension step during each cycle

issues with SYBR green

can bind to any dsDNA including primer dimers, PCR mis-anneal products, etc --> solution is to perform a melting curve

dissociation curve steps

1. all products generated during PCR amplification are melted at 95

2. temperature is dropped for complete annealing of dsDNA

3. the PCR mix is then subjected to a gradual increase in temp to 95, each increase in temperature you have partial denaturation and a decrease in fluorescence

dissociation curve results

1. a single peak means a single amplicon

2. >= 2 peaks mean it has multiple amplicons

TaqMan probe-based PCR

- probe has a fluorescent dye attached at the 5' end and a quencher at the 3' end

- relies on the 5' to 3' exonuclease activity of Taq DNA pol

- when the reporter and quencher are in close proximity--> no fluorescence

- as the polymerase extends, the 5’ to 3’ exonuclease activity of Taq DNA polymerase is also activated and it chews away any existing sequence in its path during the extension of the PCR product

- fluorescence occurs when reporter dye and quencher are no longer in close proximity

- pros of taqman probe-based pcr is that it is specific and a multiplex reaction that can be done in a single tube/well, con is that it is expensive and harder to optimize

restriction enzymes (restriction endonucleases)

an enzyme that recognizes specific sequences with DNA molecules and makes double stranded breaks at or near the recognition sites to break DNA into fragments

• found in bacteria and provide a defense mech against invading bacteriophages

  • inside a prokaryote cell, RE selectively cut up foreign DNA in a process called restriction digestion, whereas the host DNA is protected by a restriction-associated methylase that modifies the prokaryotic DNA and blocks cleavage

why do restriction enzymes exist?

they were made by bacteria as a defense mechanism against invading bacteriphages

how do host bacteria protect themselves from RE digest

- host DNA is protected by restriction-associated methylase that modifies the host DNA and prevents cleavage

- hemi-methylated sequences are the preferred substrate for the restriction-methylase so freshly replicated host DNA is more rapidly methylated than invading DNA

what components classify RE

1. cofactor requirements

2. sequence specificity

3. cleavage position

4. subunit composition

type 1 restriction enzymes

1. cofactor requirements: require S-adenosyl methione, and hydrolyze ATP for energy

2. sequence specificity : recognize specific sequences but the recognition sequences have no easily recognizable common features

3. cleavage position : cut DNA 1-5kb from the recognition sequence, exact distance varies

4. subunit composition : methylase and endonuclease are in one multi-subunit protein complex and not separable (if you try to use the RE to cut target sequence, there’s a chance your targeted sequence is also going to get protected by methylase)

example: EcoKI recognizes the sequence and binds to it, however instead of cutting the sequence it uses ATP to slide it, it then cuts around 1 to 5 kb away from recognition sequence

type 3 RE

1. cofactor requirements: require ATP

2. sequence specificity : recognize specific sequences but the recogition sites have no easily recognizable common features

3. cleavage position : ~24-28 bases away from one side of the recognition site

4. subunit composition : methylase and endonuclease are in one multi-subunit protein complex and not separable (if you try to use the RE to cut target sequence, there’s a chance your targeted sequence is also going to get protected by methylase)

type 2 RE

1. cofactor requirements: requires Mg++

2. sequence specificity : recognition sequences are typically 4-, 6-, 8-bp long and often have a two-fold axis of symmetry

3. cleavage position : cleave at defined sites close or within recognition sequence, cuts at both strands

4. subunit composition : methylase and endonuclease are physically separate entities

widely used in molecular cloning

isochizomers

different enzymes that recognize the same DNA seqence and cleave at the same site

sticky ends

staggered ends created by cleavage using restriction enzymes, can sometimes leave an overhang on one of the sides

overhang ends

5’ overhang: 5’ ends are sticking out / hovering over the 3’ end after being cleaved

3’ overhang: 3’ ends are sticking out / hovering over the 5’ end after being cleaved

neoschizomers

different enzymes that recognize the same DNA sequence but cleave at different locations in the sequence

RE frequency

4^(length of recognition site in bp)

- eg. an enzyme that recognizes a 4bp sequence, will cut every 4^4 which is every 256bp

degenerate recognition sites

example: Hinc II can cut GTPyPuAC, which means it can cut GTCGAC or GTTAAC

Hinc II can always cut Sal I sequence (GTCGAC), but Sal I cannot always cut Hinc II sequence

partial restriction digest

perform an incomplete digestion of the target DNA by stopping the reaction at different time points or using less enzyme

degenerate sites

can recognize variations of a particular sequence

how to make a degenerate enzyme

incubation conditions are not correct --> star activities (the relaxation or loss of cleavage specificity in a restriction enzyme)

restriction associated methylase

methylase adds methyl group to prevent restriction enzyme digestion

i.e. BamHI associated methylase inhibits BamHI restriction digestion

non-restriction assoicated methylase

- Dam methylase: methylates A in GATC

- Dcm methylase: methylates C in CC(A/T)GG

dam methylase inhibits ClaI (5’-ATCGAT-3’), MboI (5’-GATC-3’), but not BamHI or Sau 3A

efficiency of restriction digestion can be affected by

1. supercoil of DNA: low efficiency cleavage if supercoiled

2. nature of the flanking DNA : some RE cut poorly if close to end of DNA

3. MG++: chelation (multiple bonds) of Mg may reduce efficiency

DNA ligase major role

constructing recombinant DNA, joining free 5’ phosphate end to a free 3’ hydroxyl end through a phosphodiester bond

general rules of DNA ligases

• require proximal 3'- OH and 5’ P-group

• cannot fill gaps

• does not work on single stranded nucleic acids, however RNA ligase can do this

T4 DNA ligase

- uses ATP as energy source

- ligates nicked DNA, blunt ends and complementary sticky ends (i.e. all compatible ends)

E.coli DNA ligase

- uses NAD as energy source

- ligate nicked DNA and complementary sticky ends but not blunt ends

compatible ends

every nucleotide in the overhang of one DNA can base pair with the corresponding nucleotide in the overhang of the compatible end

however, the new sequence generated may or may not be able to get cleaved by the restriction enzymes used

alkaline phosphatase

- removes terminal monophosphates from DNA, RNA, and dNTPs

- does not remove phosphate from within the phosphodiester bond

• since they remove the terminal monophosphate, DNA ligase cannot properly function because it needs the free 5’ PO₄

T4 polynucleotide kinase

- catalyzes the transfer of a phosphate group from the y-position of ATP to the 5'OH terminus of polynucleotides

- useful for preparing DNA fragments for ligation if they don’t have 5’ phosphate

- useful for radio-labeling DNA fragments with tagged phosphates, if we have fluorescence label on the ATP, we can detect/label the DNA fragments

terminal deoxynucleotidyl transferase (TdT)

- template independent "polymerase"

- adds dNTPs to the terminal 3'OH protrusion or blunt end of ss or ds DNA

- adds whatever DNTP is given

SI nuclease

single-stranded specific endonuclease --> degrades any single stranded nucleic acid including regions within a dsDNA caused by a nick, gap, mismatch or loop

RNase H

endonuclease with little sequence specificity, degrades RNA in a duplex (hybrid) with complementary strand of DNA

does NOT digest single or double stranded DNA or RNA

DNase 1

- DNA specific endonuclease with little sequence specificity

- if Mg++ present: will nick dsDNA randomly and independently

- if Mn++ present: will nick both sides of double stranded DNA at the same site, making a double stranded break

T5 exonuclease

- dsDNA specific exonuclease and ssDNA endonuclease

• on a ssDNA, it will start in the middle and starts chewing towards the 3’ end

- cleaves linear or nicked dsDNA in 5' to 3' direction

- not active on circular or supercoiled DNA

steps of recombinant protein production

1. isolate/synthesize the target gene

2. isolated gene inserted into an expression vector

3. the vector contains elements to control gene expression and selectable markers

4. vector introduced to host cells

5. host cells cultured to promote expression

selecting an expression system

prokaryotes or mammalian cells

why are prokaryotes selected for an expression system

- pros: rapid growth, cost- effective, and high protein yield

- cons: cannot do complex post-translation modification

why are mammalian cells selected for an expression system

- pros: they can do human-like post-translational modifications

- cons: expensive and hard to cultivate in large quantities

what does a gene require to be expressed in a host system

1. promotor

2. ribosome binding sites

3. terminator

promotor

a regulatory sequence where RNA polymerase and transcription factors bind

ribosome binding sites (RBS)

a crucial nucleotide sequence in the mRNA that recruits the ribosome to initiate translation

terminator

a nucleotide sequence that ensure RNA polymerase stops synthesizes mRNA at the correct point

enhancer

- can be very far away from the gene

- can be upstream or downstream, could also be in the introns

- bound by activator proteins

RNA processing

- phosphate cap and poly-A tail added

- introns are excised

- exons are spliced together

prokaryote gene expression

transcription/translation occur in the cytoplasm and at the same time

eukaryote gene expression

transcription occurs in the nucleus and translation occurs in the cytoplasm

steps for transcription

1. initiation

2. elongation

3. termination

template strand

single strand of DNA that RNA polymerase reads during transcription to build a complementary RNA molecule

non template strand / coding sequence

same as each other (except one is dna and one is rna) b/c both are complementary to the template strand

RNA pol

- RNA synthesis is catalyzed by RNA polymerase reading a ssDNA template

- RNA pol doesn't require a primer

- polymerase activity 5' to 3'

- requires a promotor and stops at terminator

- builds a new RNA molecule through base pairing with a DNA template

inducible expression of recombinant proteins

- Some lethality-causing genes are impossible to over express for a long time

- the irreversible manipulation of gene expression often drives compensatory adaptation

- switch on and off expression of a recombinant protein quickly or modulate the level of gene expression can reveal the direct consequence of a certain genetic change

why are plasmids used for recombination

- circular DNA

- has its own replication origin, so the gene of interest can be propagated

- A selectable marker allows for the selection of successful transformations

- unique restriction site to facilitate cloning of the insert DNA

- minimum amount of non-essential DNA

ori

- DNA replication occurs here, generally high in A/T

- plasmid replicons are different from the host's chromosomal DNA but they rely on the host machinary

replication initiation for E.coli genomic DNA

1. multiple copies of DnaA bind to the ori and unwind the dsDNA

2. DnaC loads helicase and DnaB onto the replication forks

3. recruitment of the primase DnaG followed by polymerase leads to synthesis of RNA primers and genetic data

ColEI plasmid

- replication does NOT require replication initiators → relaxed control → higher copy number

- partitioning of plasmid to daughter cells is random so cells rely on the high copy number to ensure each daughter cell receives at least one

E. coli chromosome

replication is tightly controlled by unstable initiator proteins → stringent control

choosing a vector

1. understand the experiment

2. contains the appropriate MCS

3. suitable copy number

4. appropriate selection markers

vectors for generating large quantities of target DNA in E.coli

- pUC-19 copy LacZ which enable for blue/white screening fo recombinant colonies