Bio In-Class Notes (Midterm 2)

Coefficient of coincidence

- (observed double crossovers freq) / (expected double crossovers freq) - CofC = 0, complete interference and no doubles - CofC = 1, no interference and observed = expected doubles - CofC < 1, positive interference, obs < exp doubles - Cof C > 1, negative interference, obs > exp doubles

Transformation

- A method to introduce recombinant DNA into the nucleus of another cell

Principles of DNA sequencing

- A method to sequence dsDNA 1. DNA Polymerization reaction 2. Rxn spiked with poisonous ddNTPs- stops polymerization 3. Reagents required: - Template DNA - DNA Polym - Primer complimentary to target DNA - dNTPs - ddNTP (low concentration) 4. Because they all have the same 5' end, products differ based on poison position

Ampicilin

- A resistance gene (Amp^R) - Any bacteria that does not have a vector just stops growing whereas the ones with Amp^R will keep dividing

Transgene

- A transgene is an "extra" gene introduced into an organism

RNA^i

- Antisense strand base pairs to the eyeless mRNA - If we pair the antisense strand to the eyeless mRNA, it will degrade that RNA - Known as RNA^i (by base pairing that antisense to the mRNA, it will turn that region off to turn off the gene)

Core promoter elements for RNA Pol II

- BRE, TATA box, Inr (contains startpoint), DPE

Transcription

- Binding of RNA polym and local DNA unwinding - Initiation of RNA synthesis with unwound DNA - Elongation of DNA - Termination of RNA synthesis with the termination signal

Cloning a Gene in the Plasmid Vector pUC19

- Cleave vector with restriction enzyme - Cleave foreign DNA with the same restriction enzyme - Mix vector and DNA fragment under conditions that favor base pairing - Treat with DNA ligase to join DNA pieces covalently - Results in recombinant vector carrying gene of interest - Results in interrupted lacZ gene - So, this process uses polylinker, amp^R selection, lacZ (blue/white selection) which tells you whether or not there's the right DNA inserted

Combinatorial regulation by TF

- Combination of the presence of activator TF, repressor TF - All together, they can regulate genes - A single TF can regulate multiple genes - Combination of regulators to give us the correct expression of genes

cDNA

- Complementary DNA - DNA produced synthetically by reverse transcribing mRNA. - Because of eukaryotic mRNA splicing, cDNA contains no introns - just means it's a cDNA copy of mRNAs - We take advatnage of the poly A tails

Promoter and terminator signals

- Consensus sequences, which comprise the most commonly encountered nucleotides found at specific locations in DNA or RNA - In this case, RNA polym is reading these sequences from the DNA - RNA polym sees the TTGACA and TATATT (TATA box) that are promoter sequences that help it recognize that the start site is soon - Sequences (not always exactly this but similar) of GCCGCCAG and CTGGCGGC and TTTT help the RNA polym recognize where the stop site is - These are all consensus sequences, which comprise the most commonly encountered nucleotides found at specific locations in DNA or RNA

Identifying a gene associated with a disease

- Correlate inheritance of specific polymorphism with genetic disease (through genetic mapping) - Use the genome project to identify predicted and known genes in vicinity of closely-linked polymorphism - Then you could use, for example, a microarray and ask Which of the 20 genes in the neighborhood have changed expression between the ones that have or don't have the disease - So, compare total mRNAs from affected and unaffected individuals by screening microarrays (identify genes that are differentially regulated and that map close to the polymorphism- test individuals separately) - Use PCR to isolate from affected individual genomic DNA for candidate gene-- sequence to identify possible basis for the mutation - Use information to design therapy or cure (identify gene in animal model, mimic disease in model, test cure --> gene therapy!)

Chromosome interference

- Crossovers in one region decrease the probability of a second crossover close by

Generation of Recombinant DNA molecules

- Cut out DNA from two different sources with the same restriction enzyme (e.g., EcoRI), results in fragments with sticky ends - Mix fragments from both digestions and allow sticky ends of fragments to join by base pairing - Incubate with DNA ligase to link both strands covalently - This will form a recombinant DNA

Colony hybridization

- DNA probe, DNA target, determine presence within clone

Example of

- Drosophila embryos have domains of transcription factor expression - Anterior and proterior - We are using immunlocalization (need to know all these things if they rely on DNA probe, etc) to see protein products of a gene - We can see the protein products of the Bicoid, hunchback, giant, kruppel gene - Each of these would be using a different antibody to see where in the embryo is the gene being made - These TFs regulate genes expressed in more refined stripes - They work together to make a downstream gene called Eve stripe - The Eve stripe 2 (even skipped stripe) corresponds to the activator proteins present (from bicoid and hunchback) and the area where there is no repressor from the giant and kruppel - That stripe is the one place in the embryo where can turn the gene on - This is an example of reporter gene example where we are using a gene (LacZ) to report where the gene is being turned on - Allows us to determine the cis-regulatory DNA sequences to express stripe 2, w

Segmentation gene expression

- Due to specific transcription factors

Using gel electrophoresis for sequencing in cloned inserts

- Each lane corresponds to a different poison - Use a ruler and move it up one nucleotide up at a time to see which nucleotide it is - Or, in modern days, you get a graph with different colors that correspond to the different nucleotides

Eukaryotic gene activation

- Eukaryotic gene activation occurs at a distance - For eukaryotic organisms, we have enhancer sites on the DNA to which TFs bind - Those enhancer sequences can be bind to the DNA a long long way away from where transcription actually starts, but it ends up being close because the DNA is bent - The DNA is like a tether to ensure that the enhancer (where activator protein is bound to) is close to the start of transcription - The mediator is where the TF bound to the DNA interacts - TF bind to the mediator - All of these things together help determine whether the RNA pol will be able to start making DNA - The transcription complex involves the specific and general TFs

General transcription factor proteins

- Every time we turn on a gene, they result in the mRNA being made - They need to bind to the correct sequence of DNA

Zebrafish Pax6 = eyeless

- Eyeless transgene expressed in the leg

Checking the cloned insert: PCR

- Gel shows the PCR products - PCR primers base pair to the vector sequence just next to where the insertion occurs (have two primers) - These primers are represented by arrows in the slides, and the arrow pointy part is represented by the 3' end (which can have nucleotides added by DNA Polymerase, so it can get extended) - So, DNA replication reaction is carried out in PCR - The target DNA to be amplified (the template) is separated to single strands by heating (denaturing DNA) - The two primers are added to these single strands through cooling (DNA hybridization) - Now, the DNA polymerase can add dNTPs (nucleotides) to the primers to allow for DNA synthesis from primers (each strand will be extended in different directions since nucleotides always added to the 3' end) - Then repeat to form FOUR double-stranded DNA molecules - Repeat again to now produce EIGHT double stranded DNA molecules (this third cycle will result in a product with full length strands where both strands match in length)... that's the PCR product that you will see the most in all the later cycles

Enzymes involve din tryptophan biosynthesis

- Genes can be switched on and off with repressor proteins - If there are low levels of amino acid, we want the Tryp operon to be on so that we can make more enzymes to make more Tryptophan - If there are high levels of the tryptophan amino acid, so we don't need to make more amino acids - So, there is a repressor protein TF that binds to the operator sequence - When the repressor protein binds, it messes up the ability of the RNA pol to make mRNA - The tryptophan amino acid binds to the repressor protein, changing its shape, so that the repressor protein can successfully bind to the operator sequence of the DNA - BUT, if tryptophan levels are low, we won't have the tryptophan bind to the repressor, so it no longer binds to the operator, so no longer gets in the way of the RNA Pol - So the RNA Pol successfully binds and makes more mRNA to make more tryptophan

What can PCR be used for?

- Genomic template - cDNA template (copy of RNA) ---- RT-PCR (reverse transcriptase PCR) (reverse transcriptase is a virus enzyme that uses RNA as template to make a DNA copy; once the first strand is made, it turns into a regular PCR reaction in order to detect RNAs) ---- More template --> more signal - Testing cloned DNA

RNA Polymerase enzyme

- Has active site where the chemical reaction takes place (where nucleotides are added to the growing chain) - The substrates for the RNA Polym enzyme are the ribonucleoside triphosphates that are coming in through a tunnel to the active site - Newly synthesized RNA transcript leaves through the RNA exit channel - The new nucleotides are added to the 3' end one at a time to the growing chain, relying on the specificity of the base pairing

Gene expression can also be controlled by activator proteins

- If the activator protein is bound to its binding site on the DNA, it activates the transcription of mRNA - The activator protein is part of the lactose operon - The lac operon is controlled by two TF: activator (called cap) and repressor - The only situation in which the gene gets turned on is if there is activator successfully bound and no repressor bound

How is the lac operon controlled?

- If there is low glucose, a lot of lactose, we want to turn lactose into glucose, so we want the operon to be on - In this case of low glucose levels, the levels of cAMP (cyclic AMP) ligand are high and it binds to the CAP activator, changing its shape so that it can bind to the DNA - If there's no cAMP, the RNA Pol cannot bind without the help of the CAP activator - If lactose is present, it binds to the repressor protein, changes its shape, so that the repressor can no longer bond (this is good if we want to turn on the lac operon); the presence of lactose messes up the repressor protein so it can't repress anymore, thus allowing for the transcription of mRNA that will degrade lactose into glucose - Lac operon involves the activator protein AND repressor protein and both need to work together

Tryptophan operon

- In bacteria, an mRNA is made that codes for several different proteins; this mRNA is called polycistronic - How do we control whether or not to make this mRNA? We make use of the promoter - Promoter is the region in the DNA that determines whether the mRNA is made - Part of the promoter sequence is the operator, which is important for key binding operators - Together, it is called the tryptophan operon - Each of the genes involved help make the tryptophan enzymes

DNA Cloning in Bacteria using Plasmid Vector

- Insertion of DNA fragments into cloning vector - Results in recombinant plasmid containing gene of interest, containing other DNA, and nonrecombinant plasmid - Put vector into bacterial cells and collect the cells that just have the vector of interest - For many vectors, the polylinker insertion site is embedded in the bacterial lacZ gene - Will get an artificial substrate that turns blue if the lacZ gene is working - But if the insertion is working the way we want, the lacZ gene won't work, so the substrate won't turn blue - So, if the clones are blue: there's no insert; if the clones are white, there is an insert

Mass spec detects less proteins than we actually have because...

- Introns and exons

In situ hybridization; immunolocalization

- Measuring which cells express an mRNA or protein (like Drosophila embryos) - In situ hybridization: Method for looking at where mRNA is in tissues (mRNA shows blue) - Immunolocalization: antibody that binds to one protein, another antibody that binds to another protein that shows color and shows which protein works and which one doesn't (antibody probe that shows as brown) - Striped expression of segmentation genes controlling segment development - Studying gene expression provides insights into gene function

Eyeless gene

- Mutation on the eyeless gene was discovered in fruit flies - The fly with the eyeless mutation has a much smaller red eye than the wild type, so small that it's hard to see the eye at all - The eyeless transgene was expressed in the leg, so the gene was made in the wrong cells (the gene was turned on in the wrong cells)

Eukaryotic gene activator proteins can direct local alterations in chromatin structure

- One mechanism is: - Transcription regulator brings in the histone-modifying enzyme - This enzyme changes the proteins in the nucleosome in such a way that the nucleosome can open up - Another type of mechanism is that the transcription regulator (bound to DNA) recruits the chromatin-remodeling complex, which rolls around to open up the DNA and expose the TATA box - Both mechanisms open up the DNA so that DNA can be more accessible for proteins for transcription initiation (general TFs, mediator, RNA Pol)

Transcription can be controlled by proteins binding regulatory DNA sequences

- Parts of the R group of the amino can have H bond interactions on one of the DNA bases (protein binds to major groove of the DNA) - This forms a single contact between protein and DNA - There are typically 10-20 contacts - DNA-binding motifs which allow the protein to bind to specific sequences of DNA like homeodomain helix-turn-helix, zinc finger, leucine zipper motif - Sometimes the protein binds as a dimer (which can be shown in gel shift experiments) - These different motifs describe different families of proteins - In these DNA-binding motifs, protein contacts bases and sugar-phosphate DNA backbone - It's like the protein is one Velcro, the DNA is another Velcro; as long as there are enough contacts, the protein and DNA will stick together

Eyeless gene in humans and flies

- Pax6 is the human version of eyeless gene (they behave the same way) - The eyeless gene codes for the preMRA, which is then spliced into eyeless mRNA , which codes for eyeless protein, which then leads to the eyeless cDNA

Cis-regulatory sequence

- Physically attached to the gene where transcription begins

What is needed for specially designed vectors?

- Production of large amounts of a protein from a protein-coding DNA sequence cloned into an expression vector and introduced into cells

Western

- Protein electrophoresis - This uses protein for size information - Smaller proteins will migrate further down the gel (which allows us to get size information about the protein) - Gel electrophoresis can be used to separate DNA, RNA, or proteins

Western blotting or immunoblotting

- Protein with two subunits is coated with SDS to

Detecting proteins by tandem mass spectrometry

- Proteins are digested into pieces of about 10 amino acids long (using protease, which cuts proteins) - Then these fragments are separated using a chromatography column - We put a charge (ionize) on these fragments of protein - Then we essentially bash the fragments with Argon gas and cause it to break somewhere between two amino acids - Mass spectrometry graph shows m/z on the x-axis and amount of signal on the y-axis - You can use the graph to determine the size of each amino acid (ex: size of PE and size of PEP tells you the size of P), and then determine which protein is that part of, then determine whether that protein was present in the cell extract at the beginning

Principles of Western blots aka Westerns

- Proteins separated into different sizes based on their rate of movement through a gel in an electric field - Usually, proteins are denatured before separation (run as unfolded polypeptide strands) - How far a protein travels is inveersely proportional to polypeptide length - The separated proteins are then transferred to a thin membrane and "polypeptide bands" are detected with antibodies

Eyeless promoter driving GFP in transgene

- Reporter gene makes a Green Fluorescent Protein - The GFP is controlled by the eyeless gene, so it only gets turned on by the eye cells which is why only the eyes of the fruit flies were fluorescent green

Replicating and storing selected DNA molecules

- Restriction enzymes - Ligase - Vectors - Insert (use eyeless DNA sequence as insert) - Clone (once the sequence is inserted, it is called a clone) - DNA transformation into bacteria - Antibiotic resistance (selectable markers) - Polylinker (aka multiple cloning site), Lac Z - Libraries: genomic, cDNA - PCR - Expression vectors to make RNA or protein Insert into the polylinker (multiple cloning site) which has recognition sequences for restriction enzymes - Restriction enzymes can cut DNA - If bacteria have the antibiotic resistance gene, they will be resistant to the antibiotic (then they can continue dividing even in the presence of antibiotic)

Plasmids as cloning vectors

- Small circular DNA that is easily taken up by bacteria (transformed)

Deep Sequencing process

- Start with DNA copies of RNA - Add adaptor DNA sequences to the ends of these DNA strands using ligation (adaptors essentially act as primers) - The surface of the flow cell has the same primer sequences on the surface, so there can be base pairing between the adaptors and the flow cell surface (PCR is used to make hundreds of copies of one molecule of DNA in one spot, so that you get many clumps of the same-sequence DNA that landed in that part of the flow cell initially) - Once we've loaded up with a bunch of spots, we do a sequencing reaction on each of the spots - Add one base at a time to sequence and read off the colors to get the sequence - Rather than pre-loading, we make DNA copies of the RNA molecules and let individual copies of the cDNA land on the surface, then amplify each single cDNA into a cluster of the same cDNA, and then do the sequencing reaction on the spots (separating out the addition of each nucleotide from the polymerase, and as you do so, you check the color to see what color the last nucleotide added was)

General transcription factors

- TFIID binds TATA box (TFIID means Transcription Factor to D) - Once TFIID binds TATA box, the TFIIB can bind - This then allows TFIIH to bind and pry apart the ds DNA using ATP - TFIIH phosphorylates the RNA Polymerase to release it so that it can start transcribing - When TFIIH phosphorylates the RNA polymerase, it changes the shape of the RNA Pol so that capping factors, splicing factors, and polyadenylation factors can get attached (so, as RNA Is being made, these factors are added to begin RNA processing to splice the RNA as it's being made; once finished, the polyadenylation tail is added) - TATA-binding protein TBP is important because the TBP helps bend the DNA, which can open up the DNA and help other transcription factors bind DNA

How can the first step of PCR allow for heating of the DNA? Don't proteins get denatured at high temperatures?

- Tac Polymerase - Lives at very high temnperatures, so its protein has evolved so that it doesn't denature at high temperatures - This is why the DNA doesn't mind being heated up in the first step of PCR

Microarray: Measuring mRNA expression of many genes

- Take two different tissue samples and label the mRNA, which is converted to cDNA and labeled with red and green fluorochrome - They are hybridized to microarray - The different colors of the spots will tell us which tissue is expressing which gene - There can also be a mix of the colors like yellow, so the gene is expressed in both tissues - The stronger/more vibrant the color of the spot, the more mRNA was in the original tissue

RNA Processing

- The 5' cap is added to all mRNAs when they are made - This 5' cap is a guanosine and extra methyl group; this guanosine is covalently attached to the first nucleotide of the mRNA - Splicing occurs because the splicing factors are recruited - Then the PolyA tail is added (extra A-rich sequence covalently attached to the end of the mRNA) - This A-rich sequence is not coded for by the Pol, it's just added - While we're making the new mRNA using RNA Pol II, the splicing machinery is cutting out the introns - There's no splicing in bacterial cells, only in eukaryotic cells

Eyeless gene cDNA copied into vector (cloning a gene)

- The eyeless cDNA can be copied into the vector - The circle of DNA (plasmid) can be put into bacterial cells, and the cell makes thousands of copies of this circle to make a lot of protein product - This also helps store the eyeless gene

Mediator

- The mediator is a type of scaffolding protein - Scaffolding proteins bring in players for a process to one place in the cell - If all the players (e.g., proteins) are floating around in the cytoplasm, they can't work together - If we have scaffolding proteins that have binding sites for all the proteins, it helps all the protein players to congregate in the same place so that a certain process can happen - In the case of the mediator, the certain process is turning on the gene

Eukaryotic gene expression can be controlled at many different steps

- The most important part of turning the gene on/off is transcription - But gene expression also involves additional layers that can be regulated, like when we splice the RNA (that can be regulated to affect whether or not we get various products of the gene) - Also, whether the mRNA is transferred into the cytoplasm can be regulated, which can then control whether mRNA is used for protein transcription - Protein function can be controlled using post-translational modifications (interactions with other protein factors) - Essentially: do we make the products? And can we regulate the products?

How does the RNA polymerase know where to start?

- There is a start site and stop site *for transcription* - RNA polymerase has a sigma factor that's attached to it - The sigma factor helps the RNA Polymerase recognize the correct sequence on the DNA where it needs to start transcription - Once the RNA polymerase starts, the sigma factor is dissociated - Once the polymerase reaches the stop site and the polymerase is terminated and released with the completed RNA chain, the sigma factor rebinds

Genetic mapping in medicine

- Through this process, we can work out which chromosome has the gene associated with a disease and determine where on the chromosome this gene is located

Peptide mass spectrometry to detect proteins

- Trypsin digestion - Collision induced dissociation (CID) - All these fragments are ionized - These ionized fragments are analyzed in the mass spectrometry output - You match known sequences to mass spectrum peaks and based on where the peaks are, you can determine which amino acids of the proteome correspond to the protein, so you know which proteins correspond to the gene - The mass spectra is matched up to the proteins of the proteome

How can we determine whether something has one or two inserts?

- Use PCR

How is a piece of DNA cloned into a vector?

- Use a restriction enzyme (like EcoR1 which has sticky ends) on the DNA with the ORF and ligase to seal the nicks

CRISPR

- Uses the concept of base pairing - In order to edit a genome using genetic scissors, scientists can artificially construct a guide RNA, which matches the DNA code where the cut is to be made - The scissor protein is Cas9, which forms a complex with the guide RNA, which takes the scissors to the place in the genome where the cut is to be made - Researchers can allow the cell itself to repair the cut in the DNA, which will usually lead to the gene being turned off - The sequence will only be present once in 10^9 sequence - CRISPR Cas9 (enzyme) gene editing is very important in molecular biology b/c it allows you to change the sequence at will and see the consequence

Checking the cloned insert: Sequencing

- We add trace amounts of poison for each of the four nucleotides, so occasionally the DNA stops getting synthesized at certain nucleotides - The color we see will tell us which nucleotide it stopped at, so now we know the sequence (b/c it's based on the template sequence it was added on top of) - The fragments are separated by gel electrophoresis - Sequencing relies on occasional "poisoning" of strand extension - Normally, DNA polymerase adds to the 3' OH end of the DNA, but in dideoxyribonucleoside triphosphate (vs. deoxyribonucleoside triphosphate), there is no OH end (b/c it was poisoned), which is a problem for DNA polymerase b/c it now has nothing to add nucleotides to - So, a small amount of one dideoxyribonucleoside triphosphate (ddATP) is used, resulting in rare incorporation of dideoxyribonucleoside by DNA polym, which blocks further growth of the DNA molecule - This is called Sanger sequencing - Whereas for normal deoxyribonucleoside triphosphate, we use normal precursors (dATP, dCTP, dGTP, and dTTP) to make a primer for DNA polymerase to work normally using the single stranded DNA molecule

Western and Immunolocalization

- Western uses an antibody probe to target protein to determine size using gel - Immunolocalization uses antibody probe to target protein to determine tissue location -

LacZ

- encodes B-galactosidase - If the B-galactosidase is working, we get a blue colony - If the B-galactosidase is not working, we get a white colony - "Blue-white selection" - This allows us to make a transgene which allows us to observe the important function of the gene we are investigating

Checking the cloned insert: Restriction enzyme digestion

- insert some insert (e.g., 1kb)

Expression vector

A cloning vector that contains the requisite prokaryotic promoter just upstream of a restriction site where a eukaryotic gene can be inserted. - Need to use transcription, then translation - This allows us to understand the function of genes and genes associated with diseases (helps with medicine) - This expression vector is brought into bacterial cells, and then into a cell (e.g., fly cell)

Immunolocalization

Antibodies - Generated by B-cells of the immune system - Recognize foreign proteins - Bind and label the foreign protein for destruction - Primary antibody <-- secondary antibody - Secondary antibody is tagged (tag is enzyme fluorescent)

Ligation

Basic principles: - Restriction enzymes recognize specific sequence in dsDNA - Blunt or sticky ends (cohesive/overhangs) - Ligase joins ends...catalyzes bond formation

Chromatin remodeling complex vs. Histone modification

Chromatin remodeling - Causes condensed nucleosomes to be spread out into decondensed chromatin - The complex uses ATP to open up the DNA by rolling the nucleosomes around Histone modification - Various proteins like histone H3 have parts of the protein that stick out (tails) and the histone 3 tail can be post-translationally modified (e.g., phosphorylated, methylated, acetylated-- a group is added to the R group of an amino acid) - This modification influences the structure of the protein, and therefore the structure of the chromatin - Depending on the nature of the post-translational modifications for certain amino acid - If lysine 9 is methylated, it causes the protein to change shape in a way that causes heterochromatin to be favored, so gene silencing - On the other hand, if lysine 9 is acetylated (2 instead of 1 carbon group added), causes the nucleosomes to spread apart and form euchromatin to express the gene - Now, all the players can see the exposed DNA that they can now

Are crossover frequencies identical across the entire chromosome?

Crossover frequencies are not identical across the whole chromosome. There tend to be less frequencies towards the end of the chromosome.

Southern/Northern

Detection of specific RNA or DNA molecules by gel-transfer hybridization - Southern is used for DNA, Northern is used for size information - Ethidium bromide tells you if there's DNA or RNA in the gel - But if we have a probe that binds to a certain RNA sequence, it

Molecular cell biology

Genes code for mRNA, which code for proteins, which give rise to cell components, which gives rise to phenotype

TATA box

It's not always an identical, absolute sequence - Sometimes it could be TATAAA, or TAATTT, etc. as long as it's similar - TATA box is a key part of the sequence to which a specific protein binds

Microarray vs. Deep Sequencing (RNA-seq)

Microarray: - Label mRNAs - Hybridize to microarray of previously added gene sequences - Look at label signal intensities on microarray dots - Analyzing microarray data requires big data computational analysis - This data can tell us about gene networks (systems biology) Deep Sequencing - Make cDNA copies of mRNAs - Ligate adapters to cDNA (fragments) - Bind individual cDNAs to (empty) flow cell surface - PCR amplify each cDNA - Sequence each amplified cDNA (count number of spots for each sequence) Similarities: - Both create a 2d surface with a lot of spots (each spot gives information about one of the [10,000] different genes) - The more mRNA from cell extract, the more spots will be on that 2d surface Differences: - In microarray, you decide ahead of time which of the 10,000 genes are represented in each spot (so you must add some DNA to each spot to mark each gene) - In RNA-seq, you make copies (cDNAs) of RNAs, fragment the cDNAs, then add the same DNA sequence to the end of each of those fragments (adapter sequences using ligation), and each fragment is bound to the flow cell surface. Each cDNA can be sequenced so that we know what gene is on that spot.

Microarray vs. RNA-seq

Microarray: - decide ahead of time to decide that you will have ___ number of genes - Pre-loaded with DNA sequences of genes - Use probes from DNA to see if any bind to any spot RNA-seq - We don't know what genes we will see - We have a blank empty surface, and then we make spots with the DNA versions of the mRNAs from the cell extract so that we can create spots - Then we can get the sequence of the DNA version of the RNA that happen to land on each position of the 2d surface

ORF

Open reading frames don't have any stop codons - Gene ORFs are very long (100, 200 amino acids) but normally stop codons are every 20 amino acids - Telltale for genes is the absence of stop codons

Different types of RNA Polymerase in eukaryotic cells

RNA Pol I transcribes most rRNA genes RNA Pol II transcribes for protein-coding genes, miRNA genes, plus genes for some small RNAs (e.g., those in spliceosomes) RNA Pol III transcribes for tRNA genes, 5S rRNA gene, and genes for many other small RNAs Mitochondrial RNA Pol transcribes for mitochondrial RNA Chloroplast RNA transcribes chloroplast RNA

SNPs

Single nucleotide polymorphisms - single locations in a genome with variable nucleotides - Polymorphism is equivalent to an allele at a locus

Techniques for detecting different DNAs or RNAs

Southern: targets DNA to determine the size of the DNA using gel) Northern: targets RNA to determine the size - In situ hybridization (can target DNA with typically DNA probes to determine chromosome location and can target RNA to determine tissue location)

In situ hybridization to chromosomes

To locate specific genes on chromosomes - Probe for a particular gene is binding to where that gene is located on the physical chromosome

Take home message

Transcription is regulated (activated and inhibited) by a variety of proteins (or protein complexes) that bind the DNA

Challenges in molecular biology

Working with a short sequence segment - Detecting in large populations selected DNA (or RNA) molecules with selected sequences of interest - Isolating from large populations of selected DNA molecules with selected sequences (one method is PCR) - Replicating and storing selected DNA molecules Working with thousands of sequence segments - Technologies for detecting thousands of DNA (or RNA or protein) molecules with sequences of interest - Understanding gene function - Linking (disease) phenotypes to genes- which of the 20,000 genes? - Big data analytical methods are needed to understand -omic scale data DNA/RNA properties - Sequence specific base pairing- nucleic acid hybridization is the basis for many techniques - High-throughput sequencing methods are revolutionizing analysis- these methods rely on base pairing and fluorescent labeling of nucleotides

Classes of RNAs

mRNAs code for proteins rRNAs form the core of the ribosome and catalyze a protein synthesis miRNAs (micro RNAs) regulate gene expression; very short RNAs that are complementary to some of the mRNAs that we make tRNAs serve as adaptors between mRNA and amino acids during protein synthesis Other small RNAs are used in RNA splicing, telomere maintenance, and many other processes

Proteome

the entire set of proteins expressed by a given cell or group of cells