Molecular P2

DNA chemical structure

Polymer of nucleotides attached by phosphodiester bond. Nucleotides are phosphorylated nucleosides. Nucleosides comprise of ribose sugar and a nitrogen base. Adenine binds to thymine through 2 H bond, guanine and cytosine binds through 3 H bond

Nucleotide Structure

Consist of deoxyribose sugar phosphorylated on carbon 5’ and hydroxylated on carbon 3’. Nitrogen base connected through carbon 1’

Purines

Guanine and adenine. Nitrogenous base with double ring structure

Pyrimidine

Cytosine and thymine. Nitrogenous base with a single ring structure

DNA replication

New DNA strand is polymerized from 5’ to 3’, reading parent strand from 3’ to 5’ direction. Occurs in semiconservative manner. DNA in unwound by helicase and replicated by DNA polymerase on leading and lagging strand.

Leading strand

Strand where DNA replication runs in 3’ to 5’ direction in a continuous manner

Lagging strand

Strand where DNA replication runs in 3’ to 5’ in a non-continuous manner. Replication apparatus will jump a short distance then copy backwards, creating Okazaki fragments that will later on be connected by ligase

DNA polymerase

Catalyze the formation of phosphodiester bonds to extend the 3’ ends of existing DNA chains. They require a DNA template in order to determine which base to add next according to the base-pairing rules

Helicase

Unwind and untangle dsDNA

Primase

Synthesizes a short RNA to prime DNA synthesis

Methylases

Add methyl groups to nitrogen bases

Deaminases

remove amine groups from nitrogen bases

Nucleases

Cut DNA internally (endonuclease) or from the ends (exonucleases). Restriction endonucleases cut dsDNA

Ligase

Catalyze formation of a single phosphodiester bond btwn two adjacent nucleic acid fragments. Seals gaps and join two shorter pieces of DNA into one longer piece

Recombination

Mixture and assembly of new genetic combinations. Gene cloning is a method of in vitro recombination. Occurs in vivo by crossing over and then random assortment of chromosomes. Requires plasmid vector, restriction enzymes and DNA ligase for recombinant technology

Plasmids

Used to move gene from cell to cell. Occur in bacteria and most have circular chromosomes as opposed to linear in animal, fungi, plants. Efficient at xferring genes from one cell to another as opposed to fragments of DNA. Can create recombinant organisms by introducing plasmid DNA into cells in vitro

Conjugation

DNA is transferred btwn bacteria through a tube btwn cells. Two types of bacteria sex F+ and - must come in contact with each other in order to mate. Filamentous bridge develops btwn cells. F+ bacteria have fertility factor and genetic info move from + to -. Both cell becomes F+ after mating

Transduction

DNA is accidentally moved from one bacterium to another by a virus. Bacterial virus or bacteriophages infect bacterial cells. Specialized protein coats that allow virus to insert DNA through cell wall into bacterial cell wall.

Transformation

bacterium takes up a piece of DNA floating in the environment. Nonvirulent bacteria can become virulent from integrating virulent DNA fragment. Done in recombinant technology with physical insertion of DNA into bacteria.

Gene

Ordered sequence of nucleotides on a chromosome that encodes a specific functional product. Contain nucleotide sequences that will be transcribed and/or translated to protein, including structural sequence and regulatory sequence.

Messenger RNA (mRNA)

Take message from DNA to the ribosomes

Ribosomal RNA (rRNA)

Primary component of ribosomes

Transfer RNA (tRNA)

Transport amino acid to ribosomes for building proteins

Transcription

Process of copying one strand of DNA into RNA. mRNA then carries the info in DNA to ribosome to allow for translation into protein. Similar process to DNA replication but uses RNA polymerase instead

Transcription initiation

First step of RNA transcription. RNA synthesis begins at promoter codon. RNA polymerase and accessory proteins assemble on DNA at this site

Transcription elongation

Second step of transcription. RNA polymerase synthesize RNA using one strand of DNA as guide. Uracil is used instead of thymine. DNA unwound forms a transcription bubble. Does not require a primer and proceeds in 5’ to 3’. Much slower than DNA replication at about 50 to 100 base/sec

Transcription termination

Last step of transcription. Occurs differently in prokaryotes and eukaryotes. In prokaryotes, RNA polymerase and nucleotide signals can cause termination. Rho (helicase) can also inactivate elongation at Cys rich site. In eukaryotes, the poly A tail signals for termination

Constitutive

Genes that are turned on at all time. Includes enzymes that are involved in essential life processes i.e. Kreb’s cycle and glycolysis

Inducible

Genes that can be turned on and off, i.e. gene expression surrounding proteins involved in immune system

Epigenetics

Refers to changes or alterations to a person’s genome without changing the nucleotide sequence. Affects the regulation of gene expression causing activation of silencing. Can be caused by env factor or occuring normally. Changes can pass down to offspring. Examples such as DNA methylation and histone modification

DNA methylation

biological process whereby methyl groups are added to DNA molecules. Involved in genomic imprinting and X-chromosome inactivation

Genomic imprinting

Involves DNA methylation. Determined at birth, alleles from parents will be expressed or not expressed

X-chromosome inactivation

Involves DNA methylation. One of the two inherited X chromosomes in females will be inactivated - done randomly

Histone modification

Affects accessibility of DNA to bind to RNA polymerase. Genes may become inactive depending on how the DNA is wrapped around. Can modify histones in different ways such as acetylation, phosphorylation, methylation

Post transcriptional gene silencing

Type of epigenetics. Occurs in the cytoplasm. mRNA transcripts of certain genes are specifically targeted and degraded. Silenced by siRNA - double stranded RNA molecule which is non-coding.

Post-translational protein modification

Type of epigenetics. Can occur at any point of a protein’s life cycle. Proteins can becomes modified to increase stability or when needing to move different cellular compartments. Changes such as cleavage or the addition of functional groups may promote activation or maturity

Charging tRNA

1. amino acid + ATP → aminoacyl-AMP + Ppi

2. aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

Translation initiation

Small ribosomal subunit binds to IF-3 and 5’ of mRNA. This guides the AUG codon to the proper place in subunit. IF-2 and tRNA joins complex, then large ribosomal subunit binds.

Translation elongation

tRNA carrying the next amino acid binds to the growing peptide chain

Translation termination

Caused by nonsense codon or stop codon (UAA, UAG, UGA) which then stops protein synthesis

Protein translation

Formation of protein using transcribed RNA. tRNA is situated a the P site. Incoming tRNA, carrying the next aa will bind to the A site. Peptidyl transferase catalyze the formation of peptide bond, then the ribosome moves - shifting tRNA from A to P site and opening A site for the next incoming tRNA. Shift releases empty tRNA to E site for exit.

Organic DNA extraction

Cells are lysed, contents released are acidified then phenol and chloroform are added to form 2 layers. Top aqueous layer contains DNA, while bottom organic layer contains substances such as lipids. Amphilic items form a thin band btwn the two. DNA containing layer is extracted and ethyl or isopropyl alcohol is added to precipitate DNA to solid

Inorganic DNA extraction

Cells are lysed, then proteins are salted out with sodium acetate (low pH high salt sol.). DNA left over in liquid and precipitated out with isopropanol. Phenol not used as it can be corrosive.

Solid phase DNA extraction

Cells are lysed then acidified to precipitate cellular debris, leaving DNA in solution. DNA adsorbs onto a solid matrix (i.e. silica) in high salt buffer. DNA then washed with buffer and eluted in water or low salt buffer. Magnets are used if DNA were attached to beads.

DNA extraction from fixed tissue

Target cells lifted or scraped in buffer off of slide. Section is dewaxed with xylene and rehydrated with ethanol. Placed in mixture of tris buffer and proteinase K which digests proteins and lyses cell. DNA is released and can be used for testing

Mitochondrial DNA isolation

Isolated by centrifugation. Sample is homogenized, grinded on ice then centrifuged at low speed. Pellets of intact cells, nuclei and cell debris put into high speed centrifugation. Mitochondria pellets then lysed and protein contaminants removed by proteinase

mRNA isolation

Using bead or columns with single stranded oligomers of thymine or uracil to bind to polyA tail of mRNA. Unwanted materials are washed out, mRNA is eluted by breaking hydrogen bond of bead and polyA tail.

Electrophoresis

Movement of molecules by an electric current. Nucleic acid moves from the negative to positive pole. DNA is applied to gel, agarose or polyacrylamide. Migration depends on size of the molecule. Movement of molecule is impeded in gel, forming bands of molecule according to their speed of migration.

Pulse field gel electrophoresis

Very large DNA molecules are separated by this. Pulses of current applied in alternating dimension. Takes many form including Field-inversion (alternating +/- poles), Transverse alternative field, Contour-clamped homogenous electric field, and rotating gel.

Polyacrylamide gel electrophoresis (PAGE)

Gels are chemically cross-linked gels formed by polymerization of acrylamide with a crosslinking agent and polymerization catalyst. Used for very small DNA fragments, ssDNA, RNA and protein. Also used for nucleic acid sequencing, mutation analysis.

Capillary electrophoresis

Used to separate nucleic acids. Uses a thin glass (fused silica) capillary. Linear or cross-linked polyacrylamide or other linear polymers are used for sieving. Laser light excites fluorescent molecules as they pass detector and separation is based on size.

Electrophoresis buffers

Includes tris-borate EDTA, tris acetate EDTA or tris phosphate EDTA. The carry current and protects sample during electrophoresis. Buffer additive can be used to modigy sample molecule such as urea and formamide.

Horizontal gels

Usually, but not always agarose gels

Vertical gels

Usually, but not always, polyacrylamide gel

Ethidium bromide

Staining agent in electrophoresis to detect dsDNA

SYBR green or gold

Staining agent in electrophoresis to detect ss/dsDNA or RNA. Binds minor groove of dsDNA.

Silver stain

Staining agent in electrophoresis that is more sensitive for ss/dsDNA, RNA and proteins

Restriction enzymes

Cut DNA at specific sites in one of three ways. Sticky ends must match for optimal re-ligation. Blunt ends can be re-ligated with less efficiency than sticky ends. Sticky ends can be converted to blunt ends with nuclease or polymerase. Blund ends can be converted to sticky by ligating to synthetic adaptors

Type I restriction enzyme

Methylation/cleavage (3 subunits) >1000 base pairs from the binding site

Type 2 restriction enzyme

Cleavage at specific recognition site

Type 3 restriction enzyme

Methylation/cleavage (2 subunits) 24 to 26 bp from the binding site.

Restriction enzyme mapping

Digest DNA with restriction enzyme, resolve fragments by gel electrophoresis. The number of bands indicates the number of striction sites. Size of bands indicates distance btwn restriction sites

Southern blot

Detects for specific targets in genomic DNA. DNA is isolated and cut with restriction enzymes, then separated with electrophoresis. Transfer of cut DNA from gel to membrane done by capillary movement of buffer to dry paper, electric current, or suction and buffer. DNA fragments are exposed to labelled probe that is complementary to gene of interest.

Hybridization conditions

The temperature at which 50% of a nucleic acid is hybridized to its complementary strand. For 14 to 20 bp oligomers, T_m = 4° (GC) + 2° (AT)

Stringency

Describes the conditions under which hybridization takes place. Formaide, low salt and heat increases stringency

Radioactive detection

In Southern blot, after hybridization, unbound probes are rinsed off. Membrane is then exposed to autoradiography film and signal will be generated fromt he radioactive probes bound to the target

Nonradioactive detection

In Southern blot, unbound probes are rinsed off. Anti-digoxigenin Abs or streptavidin, conjugated to alkaline phosphatase is added to bind to digoxigenin or biotin probe, then unbound probes are washed away. Membrane then bathed in alkaline phosphatase substrate which produce light signal on membrane

Northern blot

No restriction digestion required. RNA is blotted to membrane. Probes are labeled antisense RNA or DNA. Internal/loading controls are required.

Western blot

Serum, cell lysate or protein is separated on SDS-PAGE. Proteins are then blotted to membranes by capillary or electrophoretic transfer. Probes are specific binding proteins, polyclonal/monoclonal Abs.

Dot/Slot blots

Used when size of target not required. DNA/RNA more quickly analyzed. Target DNA or RNA deposited directly on membrane on a dot or oblong bar (slot). Dot is useful for multiple qualitative analysis, slot is more accurate for quantification.

Reverse dot blot

Many different unlabeled established sequence are immobolized on membrane as targets. Labeled DNA, RNA or protein will be probes. Amount of sample that hybridizes is determined by signal from labelled sample.

Comparative genomic hybridization (CGH)

Type of reverse dot blots designed to test DNA. Test and reference DNA are labeled by incorporation of nucleotides covalently attached to fluorescent dyes then hybridized to the probes on a chip. Reference and DNA are labelled differently so they produce different color.

PCR primers

ssDNA containing 18 to 30 base complementary to region to be amplified. Determines the specificity of PCR reaction. Distance btwn binding sites determine size of PCR product

PCR denaturation

dsDNA denatured into two ssDNA at 94 to 96 C for several seconds to minutes. Lengthened for large DNA templates

PCR annealing

Two oligonucleotide primers will hybridize to their complementary sequences on each strand of denatured template. Happens at 50 to 70C

PCR extension

Polymerase synthesizes copy of template DNA. Add nucleotides to hybridized primers. Replicates template DNA by extending primers simultaneously on both strands of template. Optimal temp at 68 to 72 C

Misprimes

nonspecific hybridization of primers. Could be caused by primers hybridizing to one another forming primer dimers. Can become template for subsequent cycles of amplification and interfere with result interpretation.

Primer design

Avoid inter and intrastrand homologies. Tm for forward = Tm of reverse. G/C content of 20:80, avoid longer than GGGG. Product size btwn 100 to 700 bp.

Tailed primers

noncomplementary extensions added to 5’ end of primer sequence. Can carry non-DNA molecules such as fluorescent labels or other useful sequences.

Nested primer

Two pairs of primers used to amplify single target. Second pair binds slightly inside the binding site of the first. Increase the amount of amplicon in second round of amplification. Both second round products are binding within amplicon

Automated PCR and Detection

PCR with colorimetric detection of product. Samples amplified and products detected automatically after PCR reaction. Used for infectious disease applications. Real time or qPCR

Quantitative PCR (qPCR)

Standard PCR with an added probe or dye to generate a fluorescent signal from the product. Detection of signal in real time allows quantification. Performed in specialized thermal cyclers with fluorescent detection systems. Product grows in exponential fashion with lag, log, linear and stationary phase.

TaqMAN probe

Fluorescent probe that hybridize to the target btwn primer binding site and are digested when primers are extended. Releases a reporter dye from quencher. Amount of signal depends on the number of targets amplified

Fluorescence Resonance Energy Transfer (FRET) probe

Bind adjacent to one another on target. Allowing donor to transfer energy to reporter dye. The amount of signal depends on the amount of target present.

Molecular beacons

Bind target to produce signal. Reporter and quencher are on opposite ends of probe and held together by about 5 bp of homology when not bound. Probe binding to target overcomes the short-end homology, releasing reporter from quencher

Scorpion probes

Primer binding site is added to any target using 6’ tailed primer in an initial PCR reaction. An advantage is the fluorescent signal is covalently attached to the PCR product which allows for subsequent analysis.

Silent mutation

subsitution of one nucleotide with a different one that does not change the aa sequence

Conservative mutation

Mutation causing change in aa sequence, however does not drastically change protein function

Nonconservative mutation

Mutation causing replaceent with biochemically different aa. Changes chemical nature

Nonsense mutation

Mutation causing nucleotide substitution producing stop codon instead of aa codon. Terminates protein prematurely

Frameshift mutation

Insertion or deletion mutation causing a shift of nucleotide base, which affects all downstream codon.

Immunoassay

A biochemical mutation detection method. Abs that detect mutant or wild type proteins. IHC indentifies normal and mutant proteins in tissue section. Ags can be immobilized to detect the presence of Abs

High performance liquid chromatography (HPLC)

A biochemical mutation detection method. Uses solid and liquid phases to separate particles by size, charge, or chemical characteristics.

Gas chromatography

A biochemical mutation detection method. Samples are vaporized and separated in a gas phase

MALDI-TOF (Mass spec)

A biochemical mutation detection method. Matrix assisted laser desorption/ ionization time of flight mass spec is used in molecular separations. DNA variants can be detected by assessment of primer extension using ddNTPs.

Single strand conformational polymorphism (SSCP)

Nucleic acid based detection for mutation that utilizes hybridization. Scans several hundred bp that is based on instrastrand folding (changing of 1 bp affects folding). Folded strands can be resolved by size and shape. Has strict temp requirements

Melt curve analysis

Nucleic acid based detection for mutation that utilizes hybridization. Based on sequence effect on Tm. Can be performed w or w/o probes and FRET. Requires dyes specific to dsDNA. Dye or FRET will lose fluorescence due to denaturation of DNA. Detection instrument may convert melt curve to derivative of fluorescence.

Sequence specific primer PCR (SSP-PCR)

Nucleic acid based detection for mutation that utilizes sequencing. PCR primer extension requires that 3’ base of the primer is complementary to the template. Failure of amplification indicates mutation

Allelic discrimination

Nucleic acid based detection for mutation that utilizes sequencing. Uses fluorescent labelled probes and qPCR. Generates color signals for mutant or normal sequence.

Restriction fragment length polymorphism

Nucleic acid based detection for mutation that utilizes cleavage. Restriction enzyme site recognition detects presence of sequence change. Restriction enzyme will not cut due to a mutation in sequence.