MMG115 MT1 LG

Describe the mechanism of DNA replication.

• The double-stranded nature of DNA structure provides a mechanism for genetic information to be duplicated and copied from generation to generation

• During DNA synthesis, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (i.e., the template strand) guides the formation of the new strand of DNA and ensures that its nucleotide sequence is complementary to that of the template

Understand how the antiparallel and complementary nature of DNA dictates the directionality of synthesis

• A DNA molecule is composed of two antiparallel DNA strands held together by weak interactions such as hydrogen bonding and base stacking (hydrophobic interaction) forces between the two strands

• Two complementary strands of DNA are held together by hydrogen bonding and base stacking

• The phosphodiester “backbone” of DNA: Directionality

Identify and explain the specific roles of key enzymes at the replication fork

• Primase

• DNA Polymerase

• Helicase

• SSB Proteins

• Topoisomerase

• DNA Ligase

• Primase - synthesizes primer which provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand

• DNA Polymerase - synthesizes the new strands by adding nucleotides that complement each template strand

• Helicase - opens up the DNA double helix by breaking hydrogen bond between two strands of DNA and provide single template strand

• SSB Proteins - bind to single-stranded DNA at the replication fork and prevent the DNA double helix from re-annealing after helicase unwinds

• Topoisomerase - relaxes the DNA from its super coiled nature

• DNA Ligase - catalyzes the joining of ends of two DNA chains by forming phosphodiester bonds between juxtaposed 3’-OH group and 5’-Phosphate group of two adjacent DNA fragments. Joins Okazaki fragments.

Differentiate between the three enzymatic activities of E. coli DNA Polymerase I: the 5’-3’ polymerase, 5’-3’ exonuclease, and 3’-5’ exonuclease. Predict the resulting DNA sequence after these activities act on a given substrate

5’-3’ Polymerase Activity

• Requires a DNA template, a DNA or RNA primer with a 3’-OH, Mg⁺⁺, and dNTP

• C-terminal

5’-3’ Exonuclease Activity

• Requires dsDNA, Mg⁺⁺

• On when the 5’-3’ polymerase is active

• Together with the 5’-3’ polymerase activity, required for the Nick Translation reaction

  • Can be separated to generate the Klenow fragment

• N-terminal

3’-5’ Exonuclease Activity

• Proof-reading activity

• Blocked by polymerase activity

• Can trim 3’ overhangs

• C-terminal

Understand the molecular forces that drive nucleic acid hybridization.

• The process of nucleic acid renaturation or hybridization occurs when a rare random collision juxtaposes complementary nucleotide sequences on two single stranded nucleic acids, allowing the formation of a short stretch of base-paired nucleic acids (named “helix nucleation”) followed by a rapid “zippering” step. Incorrect base-pairing forms transiently and are unstable.

• Formation of a new double helix requires that both strands be in an open and unfolded conformation free of secondary structures. Therefore, hybridization reactions are performed either at high temperature (5°C-10°C below Tm) or in the presence of an organic solvent such as formamide. These conditions melt out the secondary structures that result from base-pairing within the single-stranded nucleic acids.

Evaluate how environmental factors, specifically temperature, salt concentration, and pH, could alter hybridization stringency and influence the specificity of probe binding

• These factors influence formation of new double helix

• These 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

Contrast the mechanisms used to separate DNA strands and initiate synthesis in a living cell versus a PCR tube

Proteins required for replication to proceed in living cells vs. those required for a PCR reaction

• Primase - not needed for PCR since primers are added

• DNA Polymerase

• Helicase, SSB

  • Heat provides the mechanism for strands separation

• Topoisomerase

  • Templates are usually open-ended DNA

• DNA Ligase

  • No need for ligase since each strand of the target DNA sequence is synthesized as one intact piece

Illustrate the chemical mechanism of signal generation in qPCR, differentiating between non-specific intercalation (SYBR Green) and sequence-specific hydrolysis (TaqMan probes)

• SYBR Green

  • Easy to design and optimize, cost effective

  • Reaction specificity is determined solely by the primers

  • Non-specific product can also bind SYBR green and lead to an inaccurate representation of the true product of interest

  • As temperature increases, dsDNA denatures and SYBR green dissociates, resulting in decreasing fluorescence

  • Non-specific fluorescent dyes that bind any double-stranded DNA

• TaqMan probes

  • The probe has a reporter fluorescent dye attached at the 5’ end and a quencher dye at the 3’ end

  • Rely on the 5’-3’ nuclease activity of Taq DNA polymerase to cleave the dual-labeled probe during extension of the PCR product

  • Very specific; multiplex reactions can be done in a single tube/well

  • Cons - expensive, harder to optimize

    • Sequence-specific DNA probes that are labeled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence

Understand the use of Ct values for quantification and dissociation curve (melting curve) analysis to verify reaction specificity and diagnose the presence of non-specific products or primer-dimers

• Known as the threshold cycle - the number of cycles required for any one reaction to reach the threshold fluorescence

• The value is inversely proportional to the log of the initial target concentration

  • The lower the initial concentration the more cycles will be required to reach the threshold level

• You cannot use raw Ct value as absolute final quantities

Be able to look at a double-stranded DNA sequence and identify which pair of primers will successfully “bracket” and amplify that specific region

Top Strand (Coding): 5′ - A T G C G T A C G T T A G C T A - 3′

Bottom Strand (Template): 3′ - T A C G C A T G C A A T C G A T - 5′

Identify the pair of primers…

• Forward primer - 5’ - A T G C G - 3’

  • To amplify the bottom strand

• Reverse primer - 5’ - T A G C - 3’

  • To bind to the top strand

• OVERALL PRIMER MUST BE 5’-3’

Explain the biological mechanism of bacteriophage restriction and how bacteria utilize DNA methylation to protect their own genome from self-digestion

• Restriction of phage growth was due to sequence-specific endonuclease cleavage of phage DNA by bacterial cells

• The bacteria protect their own genome from degradation by expressing restriction system-associated methylases that modify the host genomic DNA at the corresponding sites

  • Hemi (half)-methylated sequence is the preferred substrate by restriction methylase. Therefore, freshly replicated host DNA, hemi-methylated, is more rapidly methylated and protected from cleavage compared to phage DNA

Explain the key differences between Type I, II, and III restriction enzymes

• Type I

  • Cofactor Requirements

    • Requires S-adenosyl methionine as cofactor

    • Hydrolyze ATP for energy

  • Sequence specificity

    • Recognize specific sequences but the recognition sequences have no easily recognizable common features

  • Cleavage position

    • Cut DNA 1-5kb from the recognition sequence, exact distance varies from DNA molecule to molecule

  • Subunit composition

    • Methylase and endonuclease are in one multi-subunit protein complex and not separable

• Type II

  • Cofactor Requirements

    • Requires Mg⁺⁺

  • Sequence specificity

    • Recognition sequences are typically 4-, 6-, or 8- bp long. Often have a two-fold axis of rotational symmetry.

  • Cleavage position

    • Cleave at defined sites close or within recognition sequence

  • Subunit composition

    • Methylase and endonuclease are physically separate entities

• Type III

  • Cofactor Requirements

    • Require ATP as cofactor

  • Sequence specificity

    • Same as Type I

  • Cleavage position

    • Cleavage site is ~24-28 base pairs away from one side of the recognition site

  • Subunit composition

    • Same as Type I

Identify and diagram 2-fold rotational symmetry within double-standard DNA sequences

A restriction enzyme recognizes the sequence shown below which contains rotational symmetry

5’ - X X X X X X - 3’

3’ - T X G X A X - 5’

a) 5’ - A T C A T C - 3’ & 3’ - T A G T A G - 5’

b) 5’ - A G C G T A - 3’ & 3’ - T C G C A T - 5’

c) 5’ - A A C G T T - 3’ & 3’ - T T G C A A - 5’

c) 5’ - A A C G T T - 3’ & 3’ - T T G C A A - 5’

Calculate the theoretical cut frequency of a restriction enzyme based on its recognition sequence

• Hpall has a 4-base pair recognition site (CCGG) and cuts on average once every: 

  • 4⁴ = 256

• HindIII has a 6-base pair (AAGCTT) recognition site and cuts on average once every:

  • 4⁶ = 4096

• NotI has an 8-base pair recognition site (GCGGCCGC) and cuts on average once every:

  • 4⁸ = 65536

Distinguish between the molecular structures of stick ends (5’ vs. 3’ overhangs) and blunt ends produced by various restriction endonucleases

• 5’ Overhangs

  • 5’-phosphate group is exposed on the protruding strand

• 3’ Overhangs

  • 3’-hydroxyl group is exposed

• Blunt ends

  • No unpaired single-stranded nucleotides

  • Both ends possess a 5’-phosphate group

Recognize the impact of dam and dcm methylases on restriction enzyme activity and DNA digestibility

• Can inhibit certain restriction endonucleases

• dam methylase

  • Methylates A in GATC sequences

• dcm methylase

  • Methylates C in CCAGG or CCTGG sequences

Predict the compatibility of DNA ends for successful ligation and identify which combinations will regenerate or destroy specific restriction sites

• DNA ends must have matching single-stranded overhangs or both be blunt ends

• Combinations

  • Same Sticky Enzymes - Regenerated

  • Compatible Overhanges - Destroyed

  • Same Blunt Enzyme - Regenerated

  • Different Blunt Enzymes - Destroyed

Compare and contrast the biochemical requirements and applications of T4 DNA ligase versus E. coli DNA ligase

• T4 DNA Ligase

  • Uses ATP as an energy source

  • Ligate 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

  • DO NOT LIGATE BLUNT ENDS

Describe the specific activities for the following enzymes: Alkaline Phosphatase, T4 Polynucleotide Kinase, S1 Nuclease, RNase H, DNase I, T5 Exonuclease

• Alkaline Phosphatase

  • Removes terminal monophosphates from DNA, RNA, and dNTPs

  • Does not remove phosphate from within the phosphodiester bonds

• T4 Polynucleotide Kinase

  • Catalyzes the transfer of phosphate group from y position of ATP to the 5’-OH terminus of polynucleotides

  • Useful for preparing DNA fragments for ligation (if they lack 5’ phosphates)

  • Useful for radio-labeling

• S1 Nuclease

  • Single-strand-specific endonuclease - degrade single-stranded nucleic acids (DNA or RNA); this includes any single-stranded region within dsDNA caused by a nick, gap, mismatch, or loop

• RNase H

  • Endonuclease with little sequence specificity

  • Degrade RNA in a duplex with a complementary strand of DNA

    • DOES NOT DIGEST SINGLE- OR DOUBLE-STRANDED DNA OR RNA (need the combo of RNA and DNA)

• DNase I

  • DNA-specific endonuclease with little sequence specificity

  • In the presence of Mg⁺⁺, nicks each strand of ds DNA independently in a random fashion

  • In the presence of Mn⁺⁺, cleaves both strands of dsDNA at approximately the same site and generates double-stranded breaks

• T5 Exonuclease

  • dsDNA specific exonuclease and ssDNA endonuclease

  • Cleaves linear or nicked dsDNA in the 5’ to 3’ direction

  • NOT active on full circular or supercoiled dsDNA

Be able to identify key components of a transcription unit

• Promoter

  • DNA sequence that recruits RNA polymerase

• TSS

  • Transcription start site

• Terminator

  • Transcription termination (sequence)

• Each transcription unit includes a promoter, an RNA-coding region, and a terminator

Distinguish between prokaryotic gene transcription and eukaryotic gene transcription

• The organization of eukaryotic genes has additional complexity

• The coding region in eukaryotes is not continuous as in prokaryotes

  • It is interrupted with noncoding intervening sequences called introns.

• In prokaryotes, transcription occurs in the cytoplasm. Transcription and translation can occur simultaneously.

  • In eukaryotes, transcription and translation are physically separated.

Understand the molecular signatures and stages of RNA synthesis including RNA structure, RNA polymerase and the transcription cycle

• RNA Structure

  • Ribose instead of deoxyribose, and uracil instead of thymine.

  • The 2’-OH group in ribose makes RNA less stable than DNA because it is more susceptible to hydrolysis

  • The presence of the 2’-OH group also changes the phosphodiester backbone of RNA, and as a result RNA is predominantly a single-stranded molecule under physiological conditions

• RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and polymerizes the RNA nucleotides

• RNA polymerase builds a new RNA molecule through base pairing with a DNA template

  • DNA-dependent RNA-polymerase

  • Polymerizes 5’ to 3’

  • Does not need a primer

  • Requires appropriate promoter

Explain why induction is used and how the Lac-inducible system functions

• Can be used for gene expression in bacterial systems

• IPTG is a molecular mimic of allolactose. It binds to the lac repressor and releases the repressor protein from the lac operator, thereby allowing the transcription of genes downstream of the lacO sites.

• In contrast to allolactose, which the cells degrade, IPTG is a non-metabolizable analog; therefore, its concentration - and thus the level of induction - is maintained consistently over time

Contrast the molecular mechanisms of replication initiation between the E. coli genomic

• E. coli genomic DNA (DnaA-dependent)

  • Multiple copies of DnaA bind ori, unwinding dsDNA

  • DnaC loads helicase DnaB onto the two replication forks, extending the single-stranded region

  • Recruitment of the primase (DnaG) followed by E. coli Polymerase IIII and B clamp leads to synthesis of RNA primers and genomic DNA

  • DEPENDS ON THE SYNTHESIS OF REPLICATION INITIATOR PROTEIN at the beginning

• ColE1-type plasmids (RNA-primed)

  • NOT DEPENDENT on the synthesis of UNSTABLE host replication INITIATOR protein

Differentiate between “relaxed" and "stringent" replication control, explaining how copy number is regulated within the host cell.

• Relaxed = can replicate when host chromosome is not (independent)

• Stringent = strictly synchronized with host chromosomal replication; strictly requires continuous host protein synthesis and specific host initiation factors

Identify and evaluate the essential elements of laboratory plasmid vectors. Justify the inclusion of specific vector components based on their functional role in molecular cloning workflows.

• Common features of plasmids

  • Circular dsDNA

  • Unique restriction sites to facilitate cloning of insert DNA

  • Minimum amount of non-essential DNA to optimize the efficiency of cloning

  • Contains its own replication origin so the gene of interest cloned into the plasmid can be propagated

  • Contains a selectable marker gene so that bacterial cells that have not picked up the plasmids will be “weeded out” on selection growth media

• Typical molecular cloning procedures using plasmid vectors

  • Choose an appropriate plasmid vector

  • Clone the target DNA into a selected plasmid vector

  • Transform E. coli with the cloning products; grow on agar plates with selection for antibiotic resistance

  • Identify bacterium clones carrying the correct plasmid-target DNA recombinants

Explain the mechanism of blue/white screening (α-complementation) to distinguish between bacteria harboring recombinant and non-recombinant plasmid.

• Bacteria needs to be plated onto antibiotic medium that also contains X-gal. B-galactosidase hydrolyzes X-gal and turn it into an intensely blue product

• Recombinants are screened by plating onto agar plate containing X-gal and ampicillin

  • Blue colonies = non-recombinant

  • White colonies = recombinant

Describe how a modified Lac-inducible system is used to achieve controlled expression of recombinant fusion proteins.

• 1) Vectors for generating large quantities of target DNA in E. coli: e.g. pUC19, a very compact plasmid that carries a LacZ’ sequence which enables blue/white screening of recombinant DNA.

• 2) Vectors for fusion protein expression in bacteria cells

  • pGEX contains a Ptac promoter for inducible expression of GST-tagged recombinant protein, and a LacI gene which encodes for the LacI repressor that represses the Ptac promoter. Addition of the inducer IPTG inactivates the LacI repressor protein

Identify the essential elements on a plasmid to enable recombinant gene expression in mammalian cells.

• Vectors for target gene expression in mammalian cells: e.g., pcDNA3 Contains CMV (cytomegalovirus) early promoter that can be recognized by the mammalian transcription machinery, eukaryote transcription termination/polyadenylation site. The polyadenylation machinery adds polyA tail to a nascent RNA molecule, which aids transcription termination and protects mRNA from degradation.

• Neomycin resistance is used as a selection marker for plasmid DNA in mammalian cells.

Compare and contrast the workflows, advantages, and limitations of conventional restriction enzyme-dependent cloning versus Gibson Assembly.

• Conventional

  • Best suited for 1 to 2 fragments; struggles with higher multi-fragment assemblies

  • Multi-step, labor-intensive

  • Restricted by the presence or absence of specific recognition sites in the backbone and insert

  • Often leaves behind vector-derived multiple cloning site (MCS) footprints or scar amino acids/nucleotides

• Gibson

  • Can seamlessly join 2+ to 6_ fragments in one reaction

  • Single-tube, isothermal reaction

  • Completely independent of restriction sites; position is dictacted purely by PCR primer desgin. Produces a clean, scareless junction without extraneous linker or restriction site scares