Donor DNA definition and 3 types of donor DNA

Donor DNA - the DNA sequence you want to take from one source and insert into another DNA molecule 1. Genomic DNA - Taken directly from an organisms genome. EX) if you take the human gene for insulin directly from a human chromosome, you're using genomic DNA -Genomic DNA contains introns (non-coding sections within genes) that bacteria can't remove when making proteins 2. cDNA Complementary DNA - Made from using mRNA as a template by using an enzyme called reverse transcriptase - DNA -> mRNA -> cDNA - Useful for expressing eukaryotic genes in bacteria because the introns don't exist in mRNA (it just has coding information) 3. Chemically synthesized DNA - DNA sequence is artificially made in a lab (don't need to take from a cell) - Machines synthesize DNA and scientist insert it into a plasmid - Useful when the exact sequence wanted is known, when you want to modify the sequence, and when you want to make a completely new DNA sequence - No DNA template is needed (unlike cells - a strand is needed as a template)

Ori

ORI = origin of replication - specific sequence of DNA where DNA replication begins. Replication begins when an initiator protein binds to the sequence -where plasmid replication begins. It allows bacteria to make many copies of the plasmid, therefore many copies of the inserted DNA -AT base rich

Similarities and differences of Eukaryotic and Prokaryotic Replication

Differences Prokaryotes: - Occurs in cytoplasm - 1 ori - replication origin on circular chromosome - No telomeres - circular DNA termination mechanisms and DNA repair systems maintain genome stability - DNA replication can occur anytime before cell division - Faster because of less detailed checks - DNA gyrase, DNA polymerase I, II, and III - One terminus - Longer Okazaki fragments Eukaryotes: -Occurs in nucleus -Multiple ori - replication origins - along each linear chromosome (larger genome) - Have telomeres to prevent lagging strand from shortening - DNA replication occurs in S phase of the cell cycle - Slower because of more detailed checks - DNA polymerase a, b, x, y, and telomerase - Multiple terminus Similarities: Same genetic code, polymerases, semi-conservative replication, lagging and leading strand, RNA primer, Okazaki fragments

Restriction Enzymes

Recognizes certain DNA sequence and cuts through it (molecular scissors) - Used to cut sequences that are to be combined (tool for recombinant DNA technology) - Cuts end in sticky ends (strands have complementary sticky ends due to hydrogen bonding) - Obtained from different species of bacteria - Recognizes a sequence, then cuts through it. -Many different restriction enzymes (like different kinds of scissors for crafts, there are different enzymes that cut different sequences) -Have directionality and specificity

Restriction enzyme directionality and specificity

Directionality = different enzymes create different types of cuts: Sticky or blunt ends Sticky - have overhangs that can only be annealed in specific orientations due to their exposed sequences Blunt - non-specific as they lack overhangs and can anneal with any other blunt end Specificity = different enzymes produce different overhang sequences. The specificity of each enzyme determines which sequences can be joined together

Ligase

Enzyme that fuses together molecules to make recombinant DNA -Molecular glue

Polylinker (multiple cloning site)

-a short synthetic segment of DNA containing a dense sequence of recognition sites for different restriction enzymes -engineered into a plasmid (cloning vector) to facilitate the insertion of foreign DNA - Allows for you to have multiple choices of where/how to insert the DNA

2 ways to generate a DNA fragment

- Through the treatment (digestion) of DNA molecules with restriction enzymes - Using PCR technology where taq polymerase creates adenine overhangs on the fragment complementary to the U overhangs in the plasmid (no restriction enzymes needed)

Recombinant DNA

DNA molecules formed by lab methods that bring together genetic material from multiple sources - DNA created by combining 2 or more fragments from different sources.

DNA Transformation and 2 ways to transform bacteria

Putting transformed plasmid into bacteria hosts Can be done in 2 ways: -Electroporation = electric shock - Heat shock = shock with heat duh

Steps to make recombinant DNA

1. Cut out the donor DNA (DNA we want to insert) using restriction enzymes which are nucleases - Donor DNA has single stranded overhangs called sticky ends as a result of the nuclease not cutting straight 2. Cut the plasmid with same restriction enzymes - Plasmid has sticky ends as a result 3. Match sticky ends 4. Ligation - ligase glues the matching sticky ends of the donor DNA and the plasmid together to crease one continuous DNA molecule 5. Transform - introduce altered plasmid into bacterial cells and hope that it takes up the plasmid to replicate it 6. Selection - Antibiotic and blue-white selection to choose successful clones

Cloning vectors

DNA molecules capable of replicating in a host that can have foreign DNA of interest inserted - Host cells are usually bacteria - There are many vectors depending on the size of the DNA insert and what kind of cell will be the host (Eukaryote or Prokaryote)

5 cloning vectors used in Recombinant DNA technology

1. Plasmid vectors 2. Bacteriophage vectors 3. Cosmids and Fosmids 4. BAC (bacterial artificial chromosome) 5. YAC (yeast artificial chromosome)

Plasmid vectors

Small circular DNA molecules that are extrachromosomal (operate outside the chromosome) - Replicate independently from the bacterial chromosome - Can be used to carry a gene of interest into a cell (used in recombinant DNA technology) EX) Human gene -> insert into plasmid -> put plasmid into bacteria -> bacteria copies plasmid / potentially makes protein Plasmid = a tiny DNA delivery vehicle -Can be designed artificially in a lab - Used for small inserts under 1000 bases - Can't amplify DNA in Eukaryotes because Eukaryotes have linear DNA - Has polylinkers: a region with many restriction sites (with restriction enzymes) for inserting DNA - Has LacZ gene - for blue-white selection - Antibiotic resistance gene - Origin of replication (Ori) - tells the bacteria it copy the plasmid

Why does the plasmid have a polylinker (MCS)?

- The polylinker allows for multiple options for inserting DNA. - If DNA fragment and plasmid have compatible restriction sites, you can use the corresponding restriction enzyme to cut them and then join them together.

Bacteriophage vectors

-Used to clone a large insert - Harbour DNA as insert package inside a phage particle - used by a cell to kill viruses -Carry up to 20,000 bases, depending on the capsid of the phage - Have to rear both the phage and the bacteria (have to work with two systems at once, not just one in plasmids)

Cosmids and fosmid vectors

Hybrid plasmids designed to accept large fragments of DNA. Skip the hassle of rearing viruses.

BAC vectors

BAC = Bacterial artificial chromosome -Can fit and clone large fragments of DNA (500 kilobases kb) - Used for large genomes

YAC vectors

YAC = yeast artificial chromosome - Is developed to transform eukaryotic cells (used directly in eukaryotic cells because they are designed to behave like yeast chromosomes - replicate in eukaryotic yeast cell) - Allows for cloning of a lot of nucleotides that can't be put in a plasmid - too big of an insert - Used because plasmids don't replicate inside eukaryotes

Why is selection (for cloning) important?

- When you try to insert your DNA into plasmids and transform bacteria, not every plasmid will successfully contain your desired DNA. You might end up with: bacteria with no plasmid bacteria with an empty plasmid bacteria with the correct recombinant plasmid - Lots of cells are involved So you need a way to select and identify the successful ones.

First step of selection (Selecting bacteria that took up recombinant plasmid)

1st = Antibiotic resistance Purpose: To find the bacteria that received a plasmid Plasmid contains an antibiotic-resistance gene - the transformed bacteria containing antibiotic-resistance gene is grown No plasmid = no resistance = bacteria die Plasmid = resistance = bacteria live After selection: Colonies of bacteria should have a plasmid BUT: it isn't guaranteed that the plasmid has desired DNA which leads to blue-white selection - The surviving bacteria could have: an empty plasmid a recombinant plasmid containing your insert - That's why you need the second selection

Second step of selection (Selecting plasmid that contains DNA insert)

2nd = Blue-white selection Purpose: To find the plasmid that contains the DNA The plasmid contains lacZ gene and the polylinker is located within the lacZ region Normally: Functional lacZ -> b-galactosidase enzyme produced -> X-Gal gets broken down -> BLUE COLONY (plasmid is present but no insert) BUT if desired DNA is inserted to the polylinker: DNA inserted -> lacZ disrupted -> b-galactosidase enzyme is functionless -> X-Gal isn't broken down -> WHITE COLONY (plasmid is present and likely contains the inserted DNA)

Reporter Gene

a gene that gives you a visible/easily detectable signal that tells you something about what's happening

lacZ

Acts as the reporter gene because it produces a visible colour difference lacZ working -> blue lacZ disrupted -> white lacZ allows for the identification of the colonies that are likely to contain the inserted DNA easy

Taq polymerase

DNA polymerase that has heat resistance. Found in thermal vents.

PCR, its purpose and what is needed

Polymerase Chain Reaction Purpose: Provides a way to make more copies of a portion of DNA - amplifies DNA What is needed: DNA fragment that we want to copy, a buffer (contains salts and ions that keep the polymerase functioning), primers, Taq polymerase, DNA nucleotides (dNTPS), PCR bead, Mg2+

Name and describe the 3 steps of PCR

1. Denaturation. The addition of heat (95degC) to break hydrogen bonds between nucleotides, separating the two strands of the DNA molecule 2. Annealing. Specifies the region of DNA that will be copied. The separated DNA strands are cooled and joined by primers (20 bases long). The cooler temperature (50-65degC)allows for the primers to bind to the specific segment of DNA we want to amplify (make copies of) Forward primer matches a sequence on the one strand of DNA at the beginning of the region we want to amplify, and the reverse primer matches the end of the region on the other strand. Temperature is lowered, allowing hydrogen bonds to form between the primers and the template, the primers anneal to their complementary regions on the template DNA - Lots of primers are added to increase the chance that they anneal to the DNA, rather than the DNA strands annealing back together - Temp. you use depends on the specific length and sequence of primers used 3. Extension. Reaction is heated back up (72degC). Taq polymerase binds to the ends of where the primers are bound, and synthesizes a new strand in the 5' to 3' direction by adding complementary nucleotides, using the original DNA molecule strand as a template. 30 Cycles are typically ran - 1 billion copies of desired DNA

Genomic libraries

Contain the entire genome of an organism as a collection of recombinant DNA molecules in phages. Recombinant technology is used to prepare large sets of recombinant DNA molecules (vectors plus inserts) that can contain entire genomes in the form of overlapping sequences. -Allows for the storage (in a bacteriophage) and multiplication of overlapping sequences that can be used to deduce whole genome sequence through computer analysis - The process: Isolate DNA. Restriction enzymes cut the genomic DNA into many fragments that can fit in phage vectors (20 000 bases). Then the phage vector is cut using same restriction enzymes so that the sticky ends match up to the genomic DNA ends, and the sticky ends of the phage DNA and genomic DNA are ligated. That vector can then infect a bacteria to produce a plaque (a book in the library) Basically: Isolate genomic DNA - Cut genomic DNA into fragments - pack those fragments into bacteriophages - infect bacterial cells (hosts) with phages so that more phages can be produced

What is a plaque

- Zones of dead bacterial cells that are cloned bacteriophages - Have many different fragments of genes from the genome, meaning only a few contain the gene we want

What happens when a phage (with recombinant DNA) infects a bacteria?

1. Phage enters the bacterial cell 2. The phage reproduces 3. The bacterial cell is destroyed 4. New phages are released 5. New phages infect nearby bacteria 6. More bacteria destroyed and so on Eventually a clear spot appears called a plaque

cDNA library

A collection of cDNA fragments that have been cloned directly into vectors (don't need restriction enzymes to cut up cDNA because it is small due to not having introns). - The cDNA library is made up of duplicated cDNA (complementary DNA) fragments that have been inserted into a set of host cells. They form some of the transcriptomes of an organism. These fragments are stored in an “library”.

Screening

The process of finding a certain DNA sequence that contains the gene of interest

Screening process

1. Transfer DNA from plaques to a membrane to make a print of the plate 2. Positively charged membrane causes some DNA from the plaques to stick to the membrane (DNA is (-) so forces attract) making a print or copy of the petri dish 3. Add a labeled (radioactive or fluorescent) DNA probe (single stranded DNA) that is complementary to the desired gene 4. DNA-DNA hybridization - Probe binds to complementary sequence which is targeting the gene of interest 5. Autoradiograph using x-ray detect the label 6. Pick up desired gene and clone it in bacterial cells for amplification

Gel electrophoresis

Purpose: Separates DNA, RNA and Proteins based on size and charge using an electric field. Used to check if PCR product is a success, or cloning was successful DNA is (-) charged because of the phosphate group and gel electrophoresis relies on that (-) charge to separate DNA molecules based on size. Electrophoresis plate: Wells - contain DNA on one side that is (-) charged Just gel is on the other side that is (+) charged DNA travels from (-) site to (+) site

What does DNA fragment length have to do with speed in Electrophoresis

Longer DNA fragments move slower and are closer to wells Shorter DNA fragments move faster and are farther from wells, closer to the electrode

How do we determine the lengths of DNA on a gel electrophoresis plate?

A DNA ladder (marker) is a mixture of DNA fragments of known standard sizes that is put in a well, and after electrophoresis is run, it allows us to estimate the length of the fragments

Southern blotting and its process

Purpose: To detect specific DNA sequences in a sample by transferring DNA fragments into a membrane and hybridizing them with a labeled probe Process: 1. Separate DNA fragments by gel electrophoresis 2. Blot DNA from gel onto a positively charged membrane. - Soak gel in NaOH to denature DNA into single strands at room temp. - Cover with paper towel and a little weight to keep everything in place - Capillary forces will cause buffer to go through the gel and onto the paper towel, thus transferring the DNA from the gel to the membrane and preserving the relative positions of the band (we essentially made a print/copy of the plate) 3. Add labeled (fluorescent or radioactive) DNA probe that was boiled (to become single stranded) that is complementary to the target sequence 4. Hybridize the probe and the DNA by heating at a high temp. 5. Use x-ray film to get a radiogram that shows the bands that were identified by the probe

Northern blot

Purpose: Detects certain RNA sequences using RNA:RNA probe hybridization

Western blot

Purpose: Detects specific proteins with a protein probe within complex sample using anti-body based recognition.

Sanger Sequencing

AKA Dideoxy sequencing Purpose: to determine the precise order of nucleotide bases in a DNA fragment (reading a sequence of nucleotides) Process: To sequence DNA, it is synthesized in one round (single cycle). Primers are needed, and all 4 nucleotides need to be added in 4 different tubes (1 for ATGC) in each tube, DNA template added, primers, DNA polymerase. 1 nucleotide in tube is deoxynucleotide (DNA nucleotides have H instead of OH in 3') DNA strands are separated (denatured). The mixture is cooled so that a primer binds to the template DNA. DNA polymerase extends the new DNA strand by adding nucleotides. Occasionally, a fluorescently labeled ddNTP is incorporated. Because ddNTPs lack the hydroxyl group needed for further extension, DNA synthesis stops at that point . This creates many DNA fragments of different lengths, each ending with a fluorescently labeled base. The fragments are separated by size, and the fluorescent colors are detected in order to determine the DNA sequence.