Screening

1. genetic material of eukaryotic cells

2. double stranded or double helix structure

   1. directional - strands lie “head to toe”

   2. strands are antiparallel

      1. 5’ to 3’ on one strand

      2. 3’ to 5’ on another

   3. strands are complimentary to each other

3. Nucleotides: Adenine, Guanine, Cytosine, Thymine or A,C,G,T

   1. A-T

   2. G-C

4. has one fewer oxygen atom per nucleotide than RNA on the sugar base at the 2’ carbon

   
   

   
   

allows for splicing of RNA

The total number of Purines must equal to the total number of Pyrimidines

Thymine, Uracil and Guanine

1. Single stranded

2. Nucleotides: Adenine, Guanine, Cytosine, Uracil or A,C,G,U (NO THYMINE)

3. Has an additinal oxygen on the sugar base

4. typically require chemical modifications to be useable after synthesis

5. more suited for short term functions as it has low polymer stability

Adenine and Guanine

1. forms the core of ribosomes and provides the structural framwrok for ribosomes to carryout translation by catalyzing the formation of peptide bonds between amino acids

2. non-coding

1. allows for membrane integration

2. non-coding

1. fold into complex three dimensional structures

   1. Primary structure= RNA sequences from 5’ to 3’

   2. Secondary structure = short double-helical regions

   3. Tertiary structure = arrangement of double-helices and single stranded regions

1. contain information to produce protein

1. interprets the information in mRNA into a protein sequence

2. non-coding

1. encoded by 3 nucleotides or a codon

2. can be encomed by more than one codon

3. Structure

   1. Central carbon atom

   2. amino group (NH3+)

   3. carboxylate group (COO-)

   4. Side chain (denoted R) which differs in each AA

   
   

1. codon= AUG

2. start codon —> indicates where translation will start to form the protein

Codon that indicates where translation ends or where the end of a protein is

1. 3 nucleotides that encode an amino acids

   
   

1. DNA packaged into linear or circular units

2. found in the nucleus

pOwEr hOuSe oF tHe CeLl

1. Made up of Nucleotides

   1. Ex: DNA, RNA

2. store and carry information

3. joined by a phosphodiester bond between the 3’ hydroxyl group on the sugar and the phosphate attached to the 5’ hydroxyl of the next sugar

4. directional

   1. one end has 3’ hydroxl exposed and other end has 5’ phosphate exposed

   2. written 5’ to 3’

5. sugar+phosphates= sugar-phosphate backbone

1. Compromise of a base, sugar and phosphate

2. Can have additional biological functions such as energy storage of ATP and molecular transport

made of amino acids

carry out most of a cell’s functioin

amino acid sequence

1. amino acid chains start folding and binding to each other via hydrogen bonding of the peptide backbones creating repeated regular structures

2. localized regions of repeated regular structures such as beta-strands and alpha-helices

1. Secondary Structure of Protein

2. polypetide backbone froms a right-handed helix

3. hydrogen bods form between the C=) of one amino acid residue and the N-H four residues away

4. cylindrical structure wtih side chain

5. Helices are amphipathic

   1. hydrophilic on one side and hydrophobic on another side

      
      

1. secondary structure of proteins

2. formed when peptide backbones (beta strands) hydrogen bond to one another through their carbonyl and amide groups

3. slightly twisted, plargely planar structures

4. side chains protude above and below the sheet

5. can be amphipathic

6. have an antiparallel and parallel sheet

   1. Antiparallel sheets have N termini at oposite ends

   2. Paralle sheets have N termini at same end

   
   

Three dimensional folding pattern of a protein due to side chain interactions

combination of multiple polypeptides in a single protein

1. A chain of amino acids

2. Polypetide bonds between AAs form between the carboxyl group of one AA and the amino group of another AA

3. N-terminus = end of a polypetide with the exposed amino group

4. C-terminus = end of the polypeptide with exposed carboxyl

5. residues are numbers from the N-terminus

1. 2nd method for protein sequencing besides mass spec

2. each well on the sheet is filled with a different antibody for a specific protein

3. each antibody will bind to a different protein

4. in order to detect posttranslational modifications, we add duplicate antibodies of the ones already added but these have fluorescent labels

5. very expensive

1. way to study a protein

   1. Using transformation cloning by adding a epitope tag like GFP

   2. Create amino a cid polypeptide but will also make that GFP signal

   3. What it tells us

      1. tells us the location of the protein in real time via fluorescent microscope

      2. can be used to detect cocentration

      3. 
         

fats

ex) polysaccharides

1. Transformation

2. Transfection

3. Transduction

uptake of genetic material from the envrionment by bacterial (prokaryotes) cells

uptake of genetic materialf from environment by eukaryotes

using a virus to deliver genetic material to a cell

protein that hydrolyses (breaks up) DNA via water to break the hydrogen bonds

protein that hydrolyses RNA

protein that hydrolyses proteins

protein that hydrolyses lipids

1. region of DNA that controls a discrete hereditary characteristic, usually a product like a protein

2. contains the instructions to not only create a protein/product, but also controls when and where that product will be made

3. single gene can code for many different products/proteins

1. have internal membrane-bound compartments

2. compartmentalization is more complex in eukaryotes than prokaryotes —> specialized functions in certain areas

   1. ex) transcription in the nucleus while translation is in the cytosol

3. often larger than prokaryoes in cell size

4. all multicellular organisms are eukaryotic, BUT not all eukaryotes are multicellular

   1. Ex) S. verevisiae are single-celled fungi but eukaryotes

1. do not have internal membrane-boudn compartments

2. do have chromosomal DNA in nucleoid

1. have small nucleic acid genomes surrounded by a protein coat

2. cannot replicate on their own

3. must replicate within another organism using host’s cellular machinery

The physical site or location of a specific gene on a chromosome

the process by which DNA strands are broken and repaired, producing new combinations of alleles

DNA replication is the process by which the genome's DNA is copied in cells.

1. copying of DNA into RNA molecules

2. RNA polymerize synthesizes RNA from DNA

3. Regions of DNA signal RNA polymerase to start making RNA and when to stop

4. Occurs in the nucleus for eukaryotes

5. Occurs in the cytoplasm for prokaryotes

6. Steps

   1. Initiation

   2. Elongation

   3. Termination

1. RNA polymerase recognizes and binds to the promoter region of the DNA strand

2. DNA Double helix is unwound via the RNA polymerase, which includes a helicase like function for unzipping, creating the transcription bubble

3. RNA polymerase begins synthesizing RNA by adding a complementary RNA nucleotide to the template strand of DNA

   
   

1. RNA polymerase adds RNA nucleotides to the growing RNA transcript, following the template strand of DNA

2. As RNA polymerase moves along the DNA template, the double helix ahead the polymerase unwinds and the strands behind it are rewinded

3. Base pairing will occur between the RNA nucleotides and the template DNA strand, forming a single stranded RNA transcript

1. Transcription will stop when RNA polymerase reaches the terminator sequence at the end of the gene

2. RNA transcript and RNA polymerase are released from the DNA template

3. RNA transcript is modified and processed (splicing) before translation

1. enzyme that catalyzes the synthesis of RNA from a DNA template during transcription.

2. allowing the genetic information encoded in DNA to be transcribed into RNA, which can then be translated into proteins.

3. plays a regulatory role in gene expression by controlling the rate at which genes are transcribed.

4. binding of specific regulatory proteins to the promoter region, which can enhance or repress the activity of RNA polymerase

1. edits made to RNA after transcription but before translation

2. 5’ Capping

3. RNA Splicing (Alternative Splicing)

4. 3’ Polyadenylation

1. A posttranscriptional modification

2. adds an additional modified Guanine to the 5’ end of the mRNA

3. helps to protect mRNA from degration and promote translation

1. a posttranscriptional modification

2. adds a string of Adenine nucleotides to the 3’ end of mRNA

3. helps protect the mRNA from degradation and promote translation

1. posttranscriptional modification

2. introns are removed from pre-mRNA and exons are joined together to form mature mRNA

1. process by which different mRNA molecules can be produced from a single gene

2. selective splicing of exons/coding regions of a gene

3. Different combinations of exons can be spliced together to create mRNA molecules with different sequences and therefore different protein products.

4. can increase the diversity of protein products that can be generated from a single gene, thereby increasing the complexity of the proteome.

1. Turning information in mRNA into a protein

2. distinct sequences in the mRNA indicate where the translation begins and finishes

3. Stages

   1. Initiation

   2. Elongation

   3. Termination

1. A site

   1. Amino acid site

2. P site

   1. Peptide site

3. E site

   1. Exit Site

1. the first stage of translation

2. small ribosomal subunit with initiator tRNA carrying starting amino acid methionine, bound to the P Site

3. Large ribosomal unit joins the small ribosomal subunit forming the complete ribosome

1. Ribosome synthesizes the protein chain by adding amino acids one by one

2. Ribosome moves along the mRNA in the 5’ to 3’ direction

3. Reads codons and brings in the appropriate aminoacyl-tRNA molecules which will bind to the A site

4. each tRNA molecule will match to its anticodon with the complementary codon on the mRNA to ensure the correct amino acid is added to the peptide chain

5. Peptide bond is formed between the amino acid in the P site and the amino acid in the A site, removing the bond between the amino acid in the P site and its repsective tRNA molecule

6. tRNA molecule in P site shifts to the E site and is released

7. tRNA with growing polypeptide chain in the A site is shifted to the P site

8. Cycle repeats until a stop condon is encountered

1. final stage of translation

2. When the ribosome encounters a stop codon (UAA, UAG, UGA), there is no corresponding tRNA brought in

3. Instead GTP (energy) is brought in to bind to the stop codon, triggering the release of the completed polypeptide chain formed in translation by shifting the tRNA carrying the chain to the E site

4. This also triggers the dissociation of the ribosome and its subunits

1. Lyse cells to release and isolate ribosomes. Halt translations in ribosomes

2. Treat isolated ribosomes wiht RNase to digest any unprotected RNA as we want to isolate the ribosome-protected fragments which represent the position of the ribosomes on the mRNA during translation

3. Size Selection

4. Adapter Ligation: add adapter seqeunce to the RPFs

5. Reverse Trascription: convert the RPFs into cDNA

6. Circularization

7. rRNA depeletion via complementary strands and biotin

8. Library Prep and amplification

9. Sequencing

10. 
    

1. another DNA repair mechanism employed by cells to fix DNA double-strand breaks

2. does not require a homologous template for repair and is considered an error-prone repair pathway. It is the primary mechanism for repairing DSBs in mammalian cells, particularly in the G1 phase of the cell cycle when a sister chromatid is not available.

3. it can lead to the loss or addition of nucleotides at the site of repair, resulting in small insertions or deletions (indels) in the repaired DNA. These indels can cause mutations and alter the genetic information.

1. Alternative to traditional cloning methods

2. allows for the joining of multiple DNA fragments with overlapping ends.

3. DNA fragments to be assembled are mixed with DNA polymerase, DNA ligase and exonucleaase

4. overlapping ends of the DNA fragments anneal to each other, and the DNA polymerase fills in the gaps, creating a continuous DNA strand. The exonuclease then chews back any remaining overlapping regions. Finally, the DNA ligase seals the nicks in the DNA backbone, resulting in a fully assembled construct.

   
   

exchange of genetic material between two similar or identical DNA molecules. This process plays a crucial role in repairing DNA damage, ensuring proper chromosome segregation during cell division, and generating genetic diversity.

1. clustered regularly interspaced short palindoromic repeats

2. 2 Classes

3. 3 Stages

   1. Adaptation

   2. crRNA Maturation

   3. Interference

4. Uses

   1. Gene editing

   2. Gene regulation

   3. Epigeneome Editing

   4. Cromatin Imaging

   5. Base Editing

   6. RNA targeting

   7. Chromatin topology

   8. Chromatin Imaging

1. Spacer Acquisition

2. CRISPR system acquires foreign DNA sequences and incorporates them into the organism genome

3. CAS will recognize the protospacer given by the phage and integrate the protospacer into the CIRSPR array alogn with the foreign DNA given by phage

1. protospacer adjacent motif

2. 2-6 bp DNA sequence

3. recognized by the Cas Protein

1. CRISPR associated anzyme, usually nuclease or nickase

2. cleaves phosphodiester bond 3-4 bp upstream of the PAM on both strans using 2 endonucleases

1. single guide RNA

2. 5’ end of sgRNA hybridizes with the comlementary strand

3. PAM sequence must be absent in gRNA to prevent self cleavage

1. CRSIPR RNA that is complementary to the target sequence

2. binds 20bp target near PAM

3. type of gRNA

1. trans-activating CRISPR RNA

2. where CAS binds to

Nucleases are enzymes that degrade nucleic acids, either DNA or RNA.

In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages).

reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest

often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

using microscopes to view samples & objects that cannot be seen with the unaided eye

1. A target sequence is rapidly and selectively amplified

2. Steps

   1. Denaturation of template DNA

   2. Annealing of primers to template DNA

   3. Extension of primers with DNA polymerase

Primers are short sequences of DNA or RNA that are complementary to specific target sequences and are used to initiate and direct the synthesis of new strands of nucleic acid during PCR, RT-PCR, and DNA sequencing.

Typically will flank the region of interest

Modified DNA polymerase that can sustain the heat used for denaturation for PCR

used to amplify and detect RNA sequences by first converting them to complementary DNA (cDNA) using the enzyme reverse transcriptase, and then using PCR to amplify the cDNA.

1. Real time PCR used to measure the amount of DNA or RNA in a sample based on the detection and quantification of amplification products during the PCR process

2. utilizes fluorescent dyes or probes that bind to the amplified DNA or cDNA in real-time, allowing for the measurement of the amount of DNA or RNA present at each cycle of amplification.

   
   

combines RT-PCR and qPCR

technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule

First generation: Sanger

Second generation: pyrosequencing, Sequencing by synthesis

(Illumina)

Third gen: Nanopore, Single-molecule real-time sequencing

( Pacific Biosciences)

From shortest reads to Longest reads:

Illumina (50-600bp) < Sanger (400-900bp) < PacBio (60,000bp) < Nanopore (2,272,580 bp)

From highest to lowest accuracy:

Sanger (99.9%) > Illumina (99.9% but has errors due to amplification and sequencing process) > Nanopore (92 - 97%) > PacBio (87%)

From most expensive to least expensive:

Sanger > Pacbio > Nanopore > Illumina

1. Longest individual reads

2. portable and accessible

1. low throughput

1. Fast runtime

2. detects 4mC, 5mC, 6mA

1. Moderate Throughput

2. Expensive Equipment

1. Potential for high sequence yield

1. Equipment is very expensive

2. Requires high concentrations of DNA

1. Highest Accuracy

2. very applicable

1. Most expensive Sequencing

2. impractical for larger sequencing projects

3. very time consuming due to PCR

1. DNA Sample extracted and prepared for sequencing via fragmentation and size selection

2. Library Prep: fragmented DNA is ligated with adapters to create long DNA strands for sequencing

3. Loading and Sequencing: DNA loaded onto nanopore device whihc contains a mebrane with nanopores and helicase attached to the membrane. DNA strand is threaded through the nanopore while an electrical current is applied to detect chenges in current flow as each individual nucleotide passes through pore. Each change in current flow is recorded as a signal

4. Base calling: Each signal recorded during sequencing is then converted into base calls as each base has a unique current flow change

5. Quality Control: base calls are processed and analyzed to grab consensus seqeunce and make sure data is accurate and reliable

   
   

1. DNA Sample extracted and prepared for sequencing

2. SMRTbell library prep: fragmented DNA is ligated with hairpin adapters to create circular DNA molecules, called SMRTbells

3. Loading and Sequencing: SMRTbells are loaded and sequening initiated using the SMRT device. Uses fluorescence detection to capture real-time signals to generate the seqeuncing data. Each base has a unique fluorescent signal which allows the for real time sequencing

4. Base calling: Fluorescent signals are converted into base calls

5. Quality Control: base calls are processed and analyzed to grab consensus seqeunce and make sure data is accurate and reliable

1. DNA Sample extracted and prepared for sequencing via fragmentation and size selection

2. Library prep: fragmented DNA is ligated with hairpin adapters to create short DNA fragments

3. Cluster generation: DNA fragments are amplified and immobilized by adding beads to form clusters of identical copies

4. Sequencing by Synthesis: Each cluster of DNA fragments will undergoe sequencing by synthesis reaction, in which fluorescently-labeled nucleotides are added and incorporated into the growing DNA strand. When each nucleotide is added to the DNA strand, a fluorescent signal specific to that nucleotide would be emitted

5. Base calling: Fluorescent signals are converted into base calls

6. Quality Control: base calls are processed and analyzed to grab consensus seqeunce and make sure data is accurate and reliable

1. error rate of individual reads = raw accuracy of one single DNA or RNA fragment read one time

2. longer reads will have more errors than shorter reads (<1000 bp)

combining information from multiple reads, deeper coverage

variation collapsed into a single mixed sequence

Each haplotype is assembled seperately —> seperating materal and paternal inherited copies of each chromosome

1. used study the functional role of specific RNAs or to develop therapeutic interventions. RNA targeting can be used to selectively degrade target RNAs (e.g., with siRNAs) or to modulate their splicing, stability, or translation (e.g., with ASOs).

2. lyse cells, treat with protease and DNase so only RNA remains

3. target RNA type that you want

   1. mRNA: poly-A tail selection

   2. small/long ncRNA: size selection

   3. anything but riboRNA: ribo depletion

4. convert to cDNA

5. construct sequencing library

6. PCR & sequence

1. used when researchers want to focus on a subset of RNA species, such as known transcripts or specific RNA variants, rather than sequencing the entire transcriptome

2. Steps

   1. Designing RNA Capture Probes: design RNA capture probes that are complementary to the target RNA molecules of interest

   2. Hybridization of RNA Capture Probes: Hybridize the RNA capture probes to the RNA sample containing the target molecules. The capture probes will selectively bind and hybridize to the complementary target RNA molecules in the sample

      
      

   3. Capture of RNA-Probe Complexes: Capture the RNA-probe complexes using magentic beads or solid surfaces to immbolize the RNA probe complexes. Then seperates the RNA probe complexes from unbound RNA and allows us to isolate our targets of interest by washing away all unbound RNA

   4. Elution of Captured RNA: Once the RNA-probe complexes are immobilized, the captured RNA molecules need to be released or eluted for further processing which enables the recovery of the captured RNA molecules

   5. Sequencing Captured RNA

1. RNA Extraction

   1. extracting total RNA from sample

2. RNA Quality Control

   1. Isolate specific RNA species via:

      1. Poly-A selection

      2. Ribo-depletion

      3. Size Selection removes any remaining contaminants or small RNA fragments that would interfere with sequencing

3. RNA Library prep

   1. converting selected RNA into cDNA using reverse transcriptase and primers that will anneal to the polyA tails of mRNA

   2. cDNA is fragmented and adapters are ligated to ends for sequencing

4. RNA sequencing

   1. Typically Illumina is used as RNA is a typically a short molecule

   2. Which is why we do PCR amplification before sequencing in order to form identical DNA clusters for Illumina Sequencing

1. allows us to grab only mRNA’s as they are the only RNA with poly-A tails due to posttransscriptional modifications

2. Done using Poly-T magnetic beads that will bind to the poly-A tails

1. Extract Total RNA

2. Hybridze complementary biotinylated oligos

3. Bind to magnetic steptavidin beads

4. Bind beads to magnet

5. rRNA depleted RNA in spernatant

1. Total RNA extraction

2. Hybridize complmentary oligos

3. Treat with RNaseH to cleave RNA in RNA:DNA

4. Treat with DNAseI to degrade DNA oligos followed by cleanup

5. returns rRNA depleted RNA

1. Isolation of mRNA method

2. removes rRNA using specific probes which usually leaves us with ncRNA and mRNA

3. Methods

   1. Subtractive Hybridization

   2. RNaseH Degradation

Fluorescence in situ hybridization (FISH) uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope.

Antibodies as a tool...

1. you have to know the correct target (antigen)

2. Post-translation modifications usually need a different antibody to recognize each modification for the same protein.

Immunoprecipitation (IP) is a method to isolate a specific antigen from a mixture, using the antigen-antibody interaction. Antigens isolated by IP are analyzed by SDS-PAGE or Western blotting.

CRISPR is a highly precise gene editing tool

It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo

1. consists of genes and “junk” DNA

A virus is a chain of nucleic acids (DNA or RNA) which lives in a host cell, uses parts of the cellular machinery to reproduce, and releases the replicated nucleic acid chains to infect more cells.

Mass spectrometry can be used for quantitative analysis to measure how much substance is present or qualitative analysis to identify a substance or give information about the structure of a compound.

visual feature of an organism

collective DNA sequence

1. number of copies of its genome that an organism possess

2. Ex)

   1. Humans = diploid or two genome copies

   2. Yeast can be haploid (one copy) or diploid (two copies)

3. polyploidy = more than two copies of a genome

   1. having extra copies can provide backup if one copy of a gene is defective

mutant phenotype is observed and the gene that causes the phenotype is then discovered

gene of interest is disrupted and phenotype is then observed

copies of a gene that are similar but different such as wild-type and mutant genes

Need both copies of an allele to be mutated in order for the mutation to occur. If only one wild-type gene is mutated, then the other copy of the wild type gene will allow for normal function

When the wild-type gene cannot compensate for the mutant gene, meaning only one copy of the mutated gene needed for mutation to actually have an effect

1. Types

   1. point mutations

   2. insertions

   3. deletions

   4. rearrangement

2. can alter the sequence and structure of a protein

3. alter regulatory regions of a gene and change expression

4. Do not necessarily always result in disease but can increase likelihood of disease

   
   

1. preferentially allows the best-performing version of genes to persist in the population as organism with “best” gene is more likely to reproduce

   
   

1. mutations that only occur in somatic cells

2. only affect the organism itself, not subsequent generations

3. cannot be inherited

1. mutations that occur in the germline cells (reproductive cells like sperm or eggs)

2. Can be inherited and affect subsequent generations

1. mutation in a single gene that causes a disases

2. Ex) phenylkenonuria

   
   

1. mutations in several genes that cause a disease

   1. Ex) Alzheimer’s disease and diabetes

Penetrance refers to the likelihood that a clinical condition will occur when a particular genotype is present

1. single nucleotide mutations that can lead to the following effects:

   1. Missense mutations

   2. Nonsense mutations

   3. Silent mutations

1. Measures absorbance patterns in order to quantify the concentration of a specific substance in a sample.

2. Sample is placed into the sample holder and light will be passed through it

3. The detector will measure the amount of light tramsmitted through or absorbed by the sampple

4. the amount of light absorbed by the sample is proportional to the concentration of the absorbing compound in the sample

5. Example

   1. We extract the bands from the paper chromatography which is used to seperate the bases into individual solutions.

   2. then use spectrophotometer for each solution at a specific wavelength for each base to measure concentration of bases via the absoption profile

1. Used to determine the three-dimensional strcuture of molecules such as proteins and nucleic acids.

2. Used to determine DNA’s molecular structure which was a double helical structure

   1. Crystallized DNA molecules and exposed them to X-rays which scatterred off the atoms in the crystals to produce the diffraction pattern below.

      
      

mutations that eliminate the function of a gene

1. builds new DNA

   1. in 5’ -3’ direction on a new strand

   2. 3’-5’ direction on old strand

2. many versions of this protein

fixes breaks in backwards (3’-5’) segement during replication

synthesize RNA primers that are complementary to parent strand

unzips the DNA

prevents tangling of DNA during replication

prevents DNA from re-zipping during replication

1. Technique to seperate molecules by size

2. Steps

   1. Load sample mixed with loading buffer into wells of gel

   2. Electrophoresis: run an electric field to the gel where (-) is at well and (+) charge is a end of the gel.

   3. Charged molecules in the samle will move to the oppositely charged electrode. So smaller more negatively charged molecules will move to the (+) node further and faster than large (+) charged molecules which will remain closer to the wells.

   4. Staning and visualization: Seperated molecules are visualized using a staining agent, typically ethidium bromide for nucleic acids, which are visualzied under ultraviolet light

3. Inverse relationship between distance migrated and molecular size

   1. the larger the molecule, the less it will migrate across the gel

4. typically will have a marker lane or a ladder lane to act as a control

5. read bottom to top

   
   

1. Steps:

   1. Obtain a DNA template for sequencing, usually a purified plasmid or PCR product.

   2. Prepare four separate reactions, each containing a DNA primer specific to the region of interest, DNA polymerase, deoxynucleotides (dATP, dCTP, dGTP, dTTP), and a small amount of one of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP).

   3. Incubate the reactions for a short period to allow DNA synthesis to occur, with the dideoxynucleotide causing chain termination.

   4. Heat the reactions to denature the double-stranded DNA into single-stranded DNA fragments.

   5. Load the DNA fragments into a polyacrylamide gel and separate them by size using electrophoresis.

   6. Visualize the separated fragments using fluorescent or staining agent

   7. The sequence is determined by identifying the nucleotide at each position by comparing the sizes of the fragments generated in the four reactions.

2. Sanger sequencing can be used to sequence a relatively short stretch of DNA, typically up to 1000 base pairs in length.

1. Sanger is preferred over Maxam-Gilbert

2. Sanger is easier to read as it just has A,C,G,T while Maxam-Gilber has G, A+G, etc.

3. Less toxic and radioactivate materials used in Sanger

4. Sanger is more cost effective, and more accurate

5. Sanger is considered gold standard until NGS

1. Used to seperate low-molecular-mass compounds based on their distribution between the stationary and mobile phase. For example, watert will act as the stationary phase and solvent will act as the mobile phase. Components with higher solubiiity in the solvent will move faster and farther up the paper. Components with low solubility willl remain closer to the starting point of the paper.

2. Used to seperate the nucleotide bases

1. process by which a cell duplicates its DNA before cell division

2. Steps:

   1. Initiation

   2. Unwinding

   3. Priming

   4. Elongation

   5. Termination

3. Occurs in the nucleus in eukaryoptes

4. Occurs in the cytoplasm in prokaryotes

1. begins are sites called origins of replication which are recognized by initiator proteins

2. Initiator protein binds to origin of replication and seperates the strands of DNA creating the replication bubble

1. Helicase unzips the DNA

2. Single-stranded binding proteins bind to the seperated DNA strands to keep them from reannealing

1. RNA Primase adds RNA primers to the template strand of DNA, providing a free 3’ end for the DNA polymerase where we begin adding nucleotides

1. DNA polymerase moves along the template strand from 3’ to 5’ direction, synthesize the complementary DNA strand in a 5’ to 3’ direction

2. DNA polymerase will add nucleotides and proofreads and correct for any errors encounted

3. Leading strand is synthesiszed continuously while lagging strand is synthesized in short fragments called Okazaki fragments where are later joined together

1. When DNA polymerase reaches end of DNA strand, or encounters an obstacle such as another DNA stand, then replication terminates

2. Newly synthesized DNA strand is proofread and repaired by DNA polymerase to ensure accurate replication

the process by which bacterial cells take up and incorporate foreign DNA from their environment, occurring naturally or induced in the laboratory for genetic engineering purposes.

process by which bacterial DNA is transferred from one bacterium to another via a bacteriophage (a virus that infects bacteria) during infection, allowing for the exchange of genetic material and potentially acquisition of new traits.

process by which genetic material (plasmids) is transferred from one bacterial cell to another via direct cell-to-cell contact, allowing for the exchange of genetic material and potentially acquisition of new traits.

1. Take a Plasmid DNA and a chromosomal DNA or PCR fragment that we want to be cloned.

2. Use a restriction enzyme to generate complimentary ends at the recombination site of gthe plasmid to allow for insertion of the new DNA

   1. Ligate the new DNA to the newly created complementary ends to create a recombinant DNA molecule

      
      

1. DNA taken from a plasmid or bacteriophage genome that can be used to propagate an incorporated DNA sequence in a host cell

2. Can be reproduced in both prokaryoptes and eukaryotes

3. Reproduction is faster in bacteria

4. reproduction in larger quantities in eukaryotes

1. allow us to sequence new organism

2. underetand how genes are expressed and their functionality

3. create transgenic animals like GMO

4. Gene Therapy

genetic sequences introduced into cloning vectors that provide a selectable phenotype, such as antibiotic resistance, to allow for the identification and isolation of cells that have taken up and incorporated the desired DNA sequence.

1. allow for the selection of cells that have taken up a cloning vector by providing a selective advantage, such as antibiotic resistance or a fluorescent protein

2. Example: ampicillin resistance gene

1. allow for the elimination of cells that have not taken up the vector or have lost it, by conferring a lethal or inhibitory phenotype, such as sensitivity to a toxin or the loss of an essential gene

2. Example: thymidine kinase gene

used to measure promoter activity or tissue-specific expression

Ex) LacZ

Foreign DNA will be expressed

Foreign DNA will not be expressed

Foreign DNA will not be expressed

1. fluorescent activated cell sorting

2. Label Cells of interest with fluorescent dyes or antibodies which allows us to sort multiple cell populations

3. SSC tells us the granularity of the cell, i.e. how much of the cell there is

4. FSC tells us the size of the cell

5. Based on the fluorescent signals emitted by each cell, which is deteced by the machine, the cells are sorted accordingly into different populations by adding a bit of charge to each cell so that the magents will help with the sorting.

6. This way we are not sorting solely based on fluorescent color but also charge

used to determine the amino acid sequence of a polypeptide or protein.

1. possesses genes that are accessible for transcription

   1. has open active chromatin and closed inactive chromatin

1. typically DNA that is never transcribed

   1. typically just closed inactive chromatin

Has highly accessible promoters, enhancers, active genes and weakly transcribed genes (typically genes that are always active)

Made up of the following:

1. quiescent,unmarked,linker histone bound euchromatin

   1. created linker DNA holding histones together

2. facultative heterochromatin, regulated

   1. has developmentally repressed genes

3. constitutive heterochromatin, permanent

   1. centromeres, telomeres, satellites, other repeats

seperate euchromatin from heterchromatin. There maybe changes that remove the barrier which will cause genes to become closer to heterchromatin

If we look at a DNA strand, we will see that if we read it as a single line, then enhancers are very far from their promoters. However, spacially they are very close allowing of interactions

used proximity ligation and selection of target regions with primers

detects via qPCR

Used to study the three dimensional organization of the genome

Steps:

1. Crosslinking: crosslink the chromatin inside the cells by treating with formaldehyde which creates strong bonds between the proteins and nucleic acids

2. Cell Lysis amd restriction enzyme digestion: crosslinked cells are lysed to release the chromatin. Chromatin is then digested by restriction enzymes. Ends of the produced DNA fragments are marked with Biotin

3. Proximity Ligation: DNA fragments created after restriction enzyme digestion are treated DNA ligase to bring together DNA frags that were close in proximity in the nucleus. Remove terminal Biotin

4. Reverse crosslinking and DNA purification: reverse the crosslink and DNA is then purified to remove proteins and other contaminants. add linkers to the DNA ends

5. Fragmentation: DNA is then further fragmented into smaller pieces via sonciation or enzymes.

6. Library Prep

7. PCR amplification

8. Sequencing: get the sequence and map back tot he reference genome which tells us which areas are spatially close to each other and the binding sequences of the protein

9. This method does not really tell us anything about the proteins themsevles

Used to investigate protein-DNA interactions, partically the binding sites of specific proteins across the genome

Steps:

1. Crosslinking protein-DNA complexes via treatment with formaldehyde

2. Cell Lysis and chromatin fragmentation: crosslinked cells are lysed to release the chromatin and chromatin is then fragmented into small DNA fragments via sonication or enzymatic digestion

3. Immunoprecipation: Using antibodies specific to the protein of interest, we isolate and enrich the DNA fragments associated with protein of interest. Antibodies will specfically bind to specific antigens like histone tail modifications, chromatin, histones, etc. Multiple antibodies are used inthis process. Precipated using protein A/G beads or magnetic beads that interact with the specific antibody chosen to isolate the DNA

4. Reverse crosslinking and DNA purification

5. Library prep

6. PCR Amplifcation

7. Sequencing

8. Tells us which specific sequences of the DAN that the protein is binding too

Proximity Ligation and pull-down of specific protein-mediated contacts, detection by sequencing

Unlimited quantity of individual DNA segments

• Allowsustosequenceneworganism

• Understand how genes are expressed and what their function might be

• Create transgenic animals(GMO)

• Gene therapy