Recombinant DNA and Sequencing

• The material covers recombinant DNA, nucleic acid structures, and related concepts.

Next Generation DNA Sequencing

• Massively Parallel sequencing of DNAs occurs concurrently
• This sequencing can produce 1.2 billion reads of ~100 base pairs in a single experiment
• This sequencing is responsible for the genetic and genomic data explosion
• The current cost of sequencing the human genome is less than $1000
• The estimate for Illumina sequencing is ~$200 in 2024

Human Genome

• The human genome has 3.2 billion nucleotides
• The sequencing was finished 2001-2004
• Key conclusions regarding the human genome are:
• About half is various sequence repeats
• About 80% is transcribed to RNA
• About 1.2% encodes for protein
• Contains approximately 21,000 protein-encoding genes
• Only a fraction of protein-encoding genes are unique to vertebrates
• Most humans differ on average by about 1 nucleotide per 1000

Construction of Recombinant DNA Molecules

• Molecular cloning of DNA allows to amplify it and make more exact copies
• DNA is cut with restriction endonuclease to generate sticky ends
• A vector, a DNA molecule capable of carrying a foreign DNA sequence and replicating in a host cell, is similarly cut to give compatible overhangs
• The overhangs are mixed and are allowed to anneal
• Ligation is achieved with DNA ligase and ATP

Molecular Cloning

• Step I can be DNA from any source like an organism, chemically synthesized or from PCR
• E. coli is a common host
• Vectors chosen based on the insert DNA size
• Bacterial plasmids (15 kb)
• Incorporation of DNA happens into a host using: -Easy transformation -electroporation methods -Complex microinjection
• Host cell replicates the recombinant DNA

Polymerase Chain Reaction (PCR)

• PCR is a convenient way of obtaining large numbers of copies of a specific DNA sequence
• It uses two synthetic ssDNA primers which anneal to each strand at the ends of the region to be amplified
• There are 3 steps in PCR: -Heat to melt apart DNA -Cool to anneal primers -Intermediate temperature to extend the primers
• PCR is facilitated by the use of heat-stable DNA polymerase from thermophilic organisms
• Amplification of template is exponential
• Template can be dsDNA or ssDNA
• It may be present down to a single molecule
• 30 cycles yields I billion-fold amplification

iClicker Question

• If PCR amplification using human genomic DNA as template is working properly, it produces a lot of the human DNA that is in between the sequences that are complementary to the primers used

Cloned Genes in Therapeutics

• Proteins produced by genetic engineering include:
• Human insulin for diabetes
• Human growth hormone for endocrine disorders
• Erythropoietin to stimulate red blood cell production
• Colony-stimulating factors for white blood cell production and activation
• Coagulation factors IX and X for blood-clotting disorders (hemophilia)
• Tissue-type plasminogen activator for lysis of blood clots after heart attacks and stroke
• Bovine growth hormone for production of milk in cows
• Hepatitis B surface antigen for vaccination against hepatitis B

Cloned Genes in Transgenic Organisms

• Transgene is a transplanted foreign gene
• Transgenic is a multicellular organism expressing a gene from another organism
• Animals are often engineered to contain a defective gene or lacking a gene entirely, which is a gene knockout
• Genetically modified crops include golden rice
• Golden rice has genes that encode for enzymes necessary to synthesize beta-carotene

DNA Fingerprinting

• Forensic DNA testing
• Relies on DNA sequence variations (polymorphisms) among people
• Short tandem repeat (STR) sequences occur at various lengths

iClicker Question

• Given STR loci for D3S1358, vWA and FGA, Suspect 3 can be identified from the data

Objectives

• Nucleic acid structure details
• DNA Supercoiling
• Physical forces that stabilize nucleic acid structure
• Protein-DNA interactions
• Chromatin

B-DNA Structure

• B-DNA (sodium salt) is a Watson-Crick structure in hydrated form
• This is the most common biologically relevant form
• Two antiparallel polynucleotide strands are wound in a right-handed helix of 20 angstroms
• Planes of nucleotide bases occur in H-bonded pairs that are nearly perpendicular to the helix axis
• Each base pair has approximately the same width
• Canonical B-DNA helix has ~10 base pairs per helical turn and has a pitch of 34 angstroms per turn

Alternative DNA Conformations

• B-DNA (sodium salt) is the Watson-Crick structure in a hydrated form
• A-DNA consists of partially dehydrated DNA duplexes, DNA:RNA heteroduplexes and dsRNA
• A-DNA has a 20 degree tilt of base pairs
• Z-DNA has a zigzag conformation and is a left-handed helix formation that occurs in transcription due to torsion
• All DNA forms do not freely interconvert
• All DNA forms are affected by physical conditions or protein binding
• DNA has limited flexibility
• X-ray structures of B-DNA show that individual residues can depart from the average B form values

Supercoiling of DNA

• Supercoiling facilitates several biological processes -packaging of DNA -replication -transcription
• Linear and circular DNA can be in a relaxed or supercoiled shape

Supercoiling of DNA (cont 1)

• When DNA is underwound, it twists to the right to relieve strain, causing negative supercoiling
• Most naturally occurring DNA is negatively supercoiled
• Overwinding in direction of helix gives positive supercoils
• Supercoiled DNA topology is described as: -L = T + W -L = linking number, the number of times one DNA strand winds around the other -T= twist, the number of complete revolutions one strand makes around the duplex axis -Normally # of bp's divided by 10.5 -W = writhing, the number of turns duplex axis makes around the superhelix axis

Topoisomerases Alter DNA Supercoiling

• Number of coils in DNA cannot be altered without cleaving at least one of its strands
• Both prokaryotes and eukaryotes express two types of topoisomerases -Type I-create transient single-strand breaks -Type II-transient double-strand breaks

Forces that Stabilize Nucleic Acid Structure

• Hydrogen bonding of bases -Thought to be the "glue" to hold the 2 strands together -Contributes only weakly to stabilization -Bases form H-bonds to water in denatured form -Some non-Watson-Crick base pairs are possible -Occur in RNA
• Stacking interactions -Hydrophobic interactions -Van der Waals stacking interactions
• Cations Shield Repulsive Forces of Negative Charges -Divalent cations are more effective

Base Stacking Energies

• Van der Waals Interactions occur with hydrophobic interactions
• Van der Waals Interactions are enthalpically favorable
• They differ from the hydrophobic effect driving protein folding
• May be due to increased polarity of bases
• Stacking energy is sequence dependent
• G-C base pairs are more stable due to improved stacking with Nearest neighbor interactions

Terminology of dsDNA versus ssDNA

• The following terms are used for double stranded DNA: -duplex -double-stranded (dsDNA) -fully base-paired -annealed -hybridized
• Conversion of duplex DNA to single stranded DNA occurs by: -denaturation -melting -dissociation -strand separation
• The following terms are used for single stranded DNA: -simplex -ingle-stranded (ssDNA) -unpaired -melted -unhybridized
• Renaturation of single stranded DNA to form duplex DNA occurs by: -hybridization -renaturation -annealing/reannealing -strand association -duplex formation -base-pairing

Agents that Destabilize Duplex Nucleic Acid

• Duplex DNA is destabilized by: -higher temperature -low ionic strength -alkaline pH -denaturants (urea, formamide)
• Duplex DNA is stabilized by: -lower temperature -moderate or high ionic strength (cations) -neutral pH -polyvalent cations (Mg, Ca, polyamines)

Mechanisms of Stabilization/Destabilization

• Temperature -Higher temp. reduce hydrophobic base stacking interactions -Ionic strength (monovalent cations) -Low ionic strength enhances charge repulsion (destabilizes) -High ionic strength reduces charge repulsion (stabilizes)
• pH -Acidic pH removes phosphate charge repulsion (precipitates) -Neutral pH allows for normal phosphate charge repulsion and normal base pair hydrogen bonding (stabilizes) -Alkaline pH disrupts base pair hydrogen bonds (destabilizes)
• Denaturants (urea, formamide) -Increase solubility of bases (reduces stacking energy) and destabilizes duplex
• Polyvalent cations (Mg2+, Ca2+, polyamines) -Decrease charge repulsion (stabilizes duplex) – in low concs

Monitoring Denaturation and Renaturation By Hypochromicity

• Purines and pyrimidines strongly absorb UV light with peak absorbance around 260 nm • In nucleic acids, base stacking reduces UV absorbance • Upon denaturation, UV absorbance increases • Thus, dsNA is hypochromic (“less colored") versus ssNA • It is useful for monitoring the inter-conversion between ssNA and dsNA as temperature is changed • The melting temp. Tm is the temp. at the midpoint of the transition between the states • It is dependent of conditions, length and base composition of the sequence: longer and higher %GC increase Tm

RNA Structure

-RNA structure is stabilized by the same forces as DNA

-RNAs frequently contain double stranded segments -An example is 5S rRNA example with about 2/3 of bases paired

• Another example is tRNA that: -Contains several unusual H-bonding of bases -Undergoes post-transcriptionally, Modified bases