Genetics — Wave 1 (CH 9 + CH21)

What genetic material must be

1) Must contain info to make an organism, 2) must be passed down from parent to offspring, 3) must be able to be copied (to be passed from parent to offspring), 4) Must be capable of changes (phenotypic variation).

1928: Fredrick Griffith Experiment

Experimented pneumococci strains, S and R. S strain had smooth capsule and they are pathogenic, R strain could not make capsule and were not pathogenic. Putting the R strain in the heat killed S strain transformed the R strain into S strain.

Transformation

Uptake of exogenous DNA into a cell. Rare because the bacteria do not like having this done to them.

1940’s: Avery, MacLeod, Mcarty Experiment

DNA, RNA, proteins, lipids, carbohydrates are the major constituents of living cells. They used, one at a time, DNases, RNases, proteases, lipases, and carbohydrates to see if transformation happened without a certain macromolecule. The macromolecule that when removed stopped transformation, is the molecule that is genetic material. And that was DNA. Otherwise same experiment as Fredrick Griffeth’. They used antibodies and centrifuging to specifically clear out R strain to see if there is S strain.

Hershey and Chase Experiment

Provided evidence that DNA is genetic material. Used radioisotopes to distinguish DNA from proteins. 32^P labels DNA specifically, 35^S labels protein specifically. Radioactively labeled phages used to infect non radioactive E. Coli cells. They figured out that what the virus’ were injecting into the bacteria was not the protein that made up virus capsid, sheath, and tail fibers, but actually DNA. This DNA created new “baby” viruses. When centrifuging the viruses after they infected the bacteria, but before the conclusion of the lytic phase, the viruses were forced away from the bacteria. (Bacteria = heavy, virus = light). The radioactive sulfur was in the supernatant, and the radioactive phosphorus, which had to have been what the virus injected, was in the pellet.

Hershey and Chase — how do you make the radioactive virus?

Grow virus up with bacteria in the presence of a lot of radioactive methionine and amino acids, so the virus’s protein parts was labeled radioactively. Grow up bacteria with dATP with radioactively labeled phosphorus and infect the bacteria with the viruses. Sulfur and phosphorus have different radioactive profiles.

Adenine + ribose

Adenosine

Adenine + deoxyribose

Deoxyadenosine

Adenosine monophosphate

AMP

Adenosine diphosphate

ADP

Adenosine triphosphate

ATP

Thymine + deoxyribose

Thymodine

Cytosine + ribose

Cytodine

Uracil + ribose

Uradine

Linus Pauling

Discovered alpha helix structure in protein.

Rosalind Franklin + Wilking

X-diffraction to find the structure of DNA—→ diameter, # bp per turn (10), double stranded (more than one strand).

1953: Watson and Crick

Discovered helical structure of DNA. Through trial and error, built the first correct model of DNA.

X-Ray Diffraction

Used mathematics discovered by Fouyer to discover stuff about DNA from diffraction patterns look like letter X on a piece of film. Can use to find 3D structure.

Chargaff Experiment

1) pulled DNA from tissue, removed the histone proteins, and purified it. 2) He cuts nitrogenous bases off with acid, hydrolyzing, and he spots them on chromatography (TLC) and separates like nitrogenous bases. 3) He uses spectrophotographer to find out how much of A, T, G, and C and in what ratio. 4) Compared base content in DNA between different organisms. Based on molarity. Used 260 nm —> UV range.

Chargaff’s Rule

A and T in same %, and C and G in same %. Even in single stranded DNA stuff. Implications: A and T % do not have to match % of C and G. He found this out from the composition of DNA from different tissues within the same species.

DNA length/general structure

10 bp = 3.4 nm; 1 bp = 0.34 nm. 1 purine x 1 pyrmadine = 3 rings per pair total. Keeps the space between nitroenous bases uniform.

DNA structure: B DNA

Right handed ( spirals away if you look at it, turns in clockwise direction). Most common form. 2 asymmetrical grooves that certain proteins can bind to and thus interact and particular sequence of bases. Major and minor groove. Bases relative to longitudinal axis to back bone are 90 degrees.

Intermolecular Forces in DNA

A and T have 2 hydrogen bonds and C and G have 3 hydrogen bonds. The 90 degree to the back bone is because of the Van-Der-Waal’s interactions, which keeps the flattened regions of the nitrogenous bases next to one another.

Human DNA

Humans have 3.0 × 10^9 bp. DNA is 6ft in a diploid cell, and 3ft long in a haploid cell.

Z DNA

Less common, left handed, 12 bp per turn, nitrogenous bases are not 90 degrees, but they are tilted. Found first in test tube. This form is favored in high salt (high ionic) conditions with alternating purine/pyrimidine sequences (GCGCGCG), negative supercoiling, and cytosine methylation at low salt conditions. Thus, due to the last reason, Z DNA can exist in a cell. Does not have asymmetrical grooves. Might play a role in transcription and chromosome structure—it is recognized by cellular proteins and can alter chromosomal compaction. Perpendicular = orthogonal

B or Z?

DNA does not have to exist just as B or Z, but can also exist B or Z at different points in a DNA sequence.

Triple Helix — 1957 — Alexander Rich

Found first in vitro — that triplex DNA can form. In 1980, natural double stranded DNA for the first time was joined with synthetic DNA to form triplex DNA, and can thus happen in the cell. Triplex is still complementary, so that G in natural DNA bonds to C in the triplex portion, and A in natural DNA bonds to T in triplex portion. Plays role in recombination and gene inactivation.

RNA

Uracil instead of Thymine, Ribose instead of deoxyribose, less stable, typically several hundred to several thousand nucleotides, typically single stranded (Retrovirus = double stranded RNA). Whenever RNA double helices form, they are typically right handed and have 11-12 bp per turn.

RNA structure

Chimeric DNA/Recombinant DNA, biology definition

DNA from 2 different sources/species.

Gene Cloning Uses

DNA sequencing site directed mutagenesis, gene probes (southern, western, and northern blotting—think SNOW DROP), expression of cloned genes.

Chromosomal vs Vector DNA

Serves as source of DNA segment of interest vs. carrier for DNA segment that is to be cloned and can replicate independently of host chromosomal DNA.

gDNA

Genomic DNA or chromosomal DNA, whole sequence.

How to clone genes

PCR, plasmid cloning (in vivo of bacteria).

What do you do to chromosomes to replicate?

Chop them into smaller pieces using, preferably, 8 bp restriction enzymes and replicate the individual parts.

Polylinker / multiple cloning site (MCS)

Cluster of unique restriction sites. Where enzymes bind and cut DNA so that the enzyme doesn’t chop the DNA too much. Bacteria use these restriction enzymes to destroy exogenous linear DNA. Site where you insert the desired DNA sequence.

2 types of vectors

1) plasmids, 2) viral chromosome.

High copy number plasmid

Lots of a plasmid in a single bacterial cell, from 25-50 copies. Cell that carries the plasmid of interest is called host cell.

OriC

Origin of replication — unique to each species.

Viral vector

Using viruses to copy DNA via lytic cycle and infecting e. Coli — harder to break open viruses and retrieve the phage DNA than doing so would be for plasmid DNA.

Restriction enzymes

An Endonuclease can cut DNA on the inside and an exonuclease can only cut from outside. They were first discovered in bacterial cells for protection from exogenous DNA in the 1960s. They modify their DNA slightly to protect themselves from their restriction enzymes. They are often dimers and each dimer takes a specific sequence and the other dimer the other.

Restriction sites

Specific palindromic sequences that are either 4bp long, 6bp long, or 8bp long. Probability problems will consist of how often they exist — (0.25)^n, where n = how long the sequence is in bp.

Sticky vs blunt ends

Sticky ends are where 2 sources that are cut and digested can be ligated together to recreate restriction site. These often contain short, single stranded regions of DNA that can base pair with another piece of DNA with complementary sequence. Blunt ends dont have these short, single stranded regions and are harder to join together and will not recreate restriction site.

X-gal

Colorless, but when in combination with B-galactose (beta galactosidase), it creates blue dye. B galactosidase usually splits lactose into galactose and glucose and comes from lacZ gene in bacteria.

Steps in gene cloning inside bacteria

Grab some DNA, PCR amplify the cDNA after doing reverse transcriptase (PCR will result in linear DNA with blunt ends if there is good proofreading), slap a plasmid with a restriction enzyme and introduce the desired DNA into the plasmid, which will have a rare but possible chance of resulting in a recombinant plasmid (otherwise plasmid just closes in on itself even with blunt ends without any inserted DNA getting in there), smack those plasmids with some E. coli and transform some bacteria (rare but still possible to pick up ONE). Throw those bacteria into antibiotic growth plate with food, which kills all the untransformed bacteria but leaves those who uptook a plasmid. Throw some X-gal in there — if blue color happens in a colony, then those bacteria are not transformed with the recombinant plasmid, but if the result is white/colorless, then the bacteria are possibly containing the recombinant plasmid. This is due to the fact that inserting a gene into the bacteria will break the gene where it is inserted, so that the bacteria with inserted plasmid cannot actually make the B-galactose to turn the X-gal blue. Doesnt always mean that it is desired recombinant plasmid, it could be random DNA—check with gel electrophoresis. Bacteria divid every 20 minutes and can duplicate sequences that aren’t even genes, like telomeres or centromeres—to use the bacteria to transcribe and translate the DNA into a protein, however, you have to actually have a promotor and cDNA without introns and a terminator for this to actually work. To replicate, there also needs to be an origin of replication.

Recombinant vector

Vector with insert.

Transfection

Putting plasmids into mammalian cells (if it were to happen, they would use cancer cells bec its easier). Also can refer to virus going into host cell.

IPTG

Lactose analogue (lookalike) that is an inducer for the promoter for the lac Z gene

Reverse Transcriptase

Clone DNA from RNA being a template. Used by retroviruses to copy their RNA genome into DNA for infection. Complementary DNA is needed to be able to remove introns so bacteria can actually translate eukaryotic genes after being put into the bacteria. This is also a DNA polymerase

Making cDNA

Get mRNA, add reverse transcriptase and dNTPs as well as a oligo-dT primer for 1st strand cDNA synthesis. The oligo dT primer is a 15 bp piece of DNA that acts as a primer. Then add RNase to digest mRNA into little primers. Then add DNA polymerase I and more dNTPS —> remove RNA and replace with DNA—uses cDNA as template, which will result in blunt ends and double stranded DNA. Then, to make the DNA safe for storage, put the cDNA into plasmids via restriction sites and whatever. Due to alternative splicing and different mRNAs being produced in the cell to act as template for reverse transcriptase, there will be multiple types of cDNA being produced, and the one that is amplified the most is the one that is in greatest quantity made by the cell and thus probably the most important.

DNA library

Can be either cDNA or gDNA. Plasmids that have the gene of interest.

RT-PCR vs RT-qPCR

Reverse Transcriptase PCR is fastest way to get cDNA in lab, while Reverse Transcriptase quantitation PCR is where you see whatever gene amplifies faster and thus is in higher quantity.

Plating the library

You get the plasmids in the DNA library and you transform bacteria on the food source + antibiotics, which will sort all the DNA for ya as well.

Screening the library

Add a piece of nitrocellulose on top of the colony groups of transformed bacteria, which will transfer some bacteria on the nitrocellulose, and soak the paper in NaOH/SDS to lyse the bacteria and denature the DNA into single strands instead of dsDNA. You can use a radioactive probe from cDNA that is single stranded to go and bind to the respective genomic DNA in the library to thus find the promoter. You then use autoradiographing on a piece of film to see the results.

PCR: General info

Developed by Kary Mullis in 1985, can let you copy DNA without needing vectors and host cells, and requires enough of the gene of interest to be known have sequence of 2 short primers. Amplification is 2^n, where n is how many cycles you run.

Steps of PCR

1) Denaturation at 94 degrees C to break dsDNA into single stranded DNA, 2) Annealing of primer at 52-65 degrees celsius to have the primer dock to the DNA, and 3) Extension at 72 degrees C to extend the DNA and finish 1 cycle. Too many cycles cannot work so maximum you can do is 40. uses Taq polymerase. Each cycle is called denaturing-annealing synthesis.

Step 2 of PCR — primer annealing and temperature

Most critical parameter. Tm is the melting point of the primer, and if the temperature is too high, the primer would bind too nonspecifically, it needs to be at the just right temperature. The primer melting point is determined by 2 things — CG bp and how long it is. Longer + more CG bp = more hydrogen bonds = higher mp, and vice versa.

PCR — Starting Materials

Requires Template DNA, oligonucleotide primers (15-30 bp long) and coplementary to sequences at the ends of the DNA fragments that need to be amplified, Taq polymerase (thermostable DNA polymerase, sweet spot of replication is 500-1500 nucleotides long)), and thermocycler.

Guanine + ribose

Guanosine

Guanine + deoxyribose

Deoxyguanosine