Pooppppppppppppppppyy exam 3

day 1 -2 of infromation

what is a gene? a gene is a region of dna that has ifnromation of a particular rna or protein. DNA contains gtenes and encode gene products and then these products carry out essential functions . like begets like - 1800 gegro medel - heritbale traits are controlled by discrete factors - tehse factors are passed from one generation to another and these factors are called genes. he worked with the pea plant - he identified simple traits he could follow over generations such as the flower color or pea shape. some pea plants are pue breeding for these traits. purple flower x purple flower = purple flower all off springs. or true breedign if a pea plant is displaying a certain trait and is matied to itself all of its descendents will deisplay the same triat. if we take that pure preeding and cross it with a pure preeding plant that makes white flowers -

so in mendels experiment he did a monohybrid cross where he crossed a round pea to a wrinkled pea - first gerneation all of the peas were round. F1 fillial generation - if he took those generation round peas and crossed them to eachother he got both round and wrinkled of generation 2. The ration of 3;1 of round to wrinkles peas. inheritance follows predictable rules.

the inhertance of traits is controlled by factors genes that occur in pairs. pea plants like humand are diploids and every chromosome is represented twice… 2n. for every chromsome pair 23 , if gene a is one then there is another gene a in the other making a pair. Genes exsist in forms called alleles. R or r. different alleles are paires one member of the pair is dominant to the other so R is dominant over the r. Any two indivuals with the same phenotype could have different genotypes. So round can have Rr or RR. homozygous or heterozygous for either allele.

module 21C

genes exxist in different alleles. two alleles must segrate during gamete formation so a big r and little r must segrgate during gamete formation . a gamete when made will pick up one gene copy or one allele. One a male gamete is produce it only picks up one gene copy or allele. when its female it will pick up one the organism will have two . genes arer in chromosomes. Chromsomes gametes is 1n. fmela ie 1n male is 1n and the diploid is 2n gamete. meiosis. this process is where the 1N or halpoid gametes will produce the diploid or 2norganism. so during mitosis is thhe distributing chormsome during the lifepsane of the organism - this is hapenning in all cell divisions in ur body .

homologous chromsomes - one from dad one from mom and then it will replicaate where it will then split and then the daughter cell will have one mom one dad. look at mitosis image. mitosis accounds for all cell division in body. In mitosis the chromsomal dna is divded between daughter cells. during meiosis is the process is chromosme being distrubuting through genreations these will produce games. So the cell will have one mom one dad, then it will replate so two mom two dad, durinng meosis one it will lead to two dad in one daughter and two mom in another cell. then it will go through meosis 2 where it will seperate so two daughter will have one dad and two will have one mom - look at meiosis.

alleles will segrgate indepently of eachothher during gamete formation so this means tha half of the female gametes will carry on allele and the other half will carry the other allele. ½ of the male will carry one and the other ½ will carry the other allele.

probability of inheriting two dominate allel is 1.2 × 1.2 = 1/4.

two recessive is also 1/4.

probability of inheriting one dominant and one recessive allele is ¼ + ¼ = ½

dihibrid cross - RRYY x rryy f2 = 9,3,3,1

yellow round x green wrinkles

canvas

module 21 c start over -

mendel -fuck ass mendel - genes occur in pairs and exsisten in different form and one allele can be dominant over the other one.

two forms of a gene pair must segragate during gamete formation . so Rr must serpeate. when an organism is made it will pick up two gene copies or two alleles. One from the female and one from the male . so RR and rr will make a Rr because it will pick up one from one parent and one from the other parent. the egg will give one and sperm will give one. Genes are in chromsoomes. this is chormosome behaviors. chromsome number of gametes is 1n and the male gamete is 1n to make 2n, So when making a baby one gene comes from the dad and one gene comes from the mom to make a diploid chromsome baby. the sperm and egg are both haploid which carries on set of the chromsome. so 23 is onne set and another set is 23 to make 46. this is meiosis. 1n or haploid areproduced from 2n organisms. mitosis is the process is distrubution chromsomes during the life spam. So mitosis will make genetically identical cells.

so one chromsome comes from dad and another from mom. They will replicate and then split off and the daughter cells will have the same one mom one dad. so it is 2n.

meosis results in 1n. because it will start wth one mom one dad in a cell. it will replicate to lead to two dad two mom and then split off where it will lead to two dad two mom in two cells and then split off again where two cells will have two mom and two will have dad. so half will be dad and half will have mom, meosis results in two divisions . so it leads to four daughter cells not identical.

half of the female gametes will care one allele and the other will carry the other allele and same thing for the male.

21D….

genetic screen -

muller discovered that exposing tissue to x-ray damages the DNa and induces mutation - certain chemicals also cause mutations. This allows for new variants and genetic alleles. genetic screen - organism is exeposed to radiation or chemcials to produce a mutagensis

they display mutant pehnotype. if the progency that inherited the mutation is isolated then the mutation is isoalted and mapped to a gene and the gene is studied using that mutation . use a wild type or normal organism, then mutagensis to create a mutation. mutageneis is eseentially random so every chromsome will experience its unique set of mtuation. ina screen you are concerd with mutations in the germ line reprroductive organs that under meoisis to produce gametes that can be passed off. so when a parent is mutagenzied, each gamete ccan carry a different mutation. and the offspring will be heterozygous for those mutations . so when amutated indivual is mated with a wild type then the offfspring will be heterozygous . greater than 90% of the genetic mutations produced are recessive to the wild type . so the wild type will hide it. so in heterozygous will not be able to see the mutation . the hetoerzygous mtuatnt must be bred to produce a homozygous recessive to see the mtuation and then identify it and isolate it .

genetic screen in a lab - we take a wild type and mutate it and then cross it with a wild type. then the offspring will have a heterozygous mutation, cross it with another wild type and the offsprings again will have a hterozyggous. But crossing those off springs iwth another heterozygous willl lead to poping up of the mutation .this heterozyggote must be bred through two generations to produce fush that are homozygous for the mutation and show a mutatnt phenotype . u have to let the siblings to mate to allow for homozygous mutationn recessive. the mutatio will lead to loss of cuntion mutations of a gene. so the mtuation of a phenotype can tell u somethign about the normal function of a gene this is because if the stripes of a zebra fish are gone then that means that the gene that was mutated is the one that gives it its striped colors so it was that gene that was affected.

genetic screens - zebra phis or the fuck ass filies.

22c

PCR is a way to synthesize and amplify without having to cone cells. PCR is fast it is used to amplify a single DNA sequence from a dilute or impure sample. It is used to analyze blood or tissue traces left behin at crime scenes. It is used for diagnostics in clinic. DNA can be snthesized in a test tube VITRO by annealing a complemetary to a single stranded DNA template strand . So the complentary primer will bind to the template strand . Once addding free nucleotdies and dna pol then the 3’ end of the primer will be extended to make a new dna strand .

ONE cycle

we have our target dna template. So first we will denature the the double stranded DNA in order to get access to the template strand. Denaturing is in the prescence of dna polymerase, dNTPS, and 2 DNA primers complementary to oppsoite strands of the target DNA. Thyen it is cooled back down in order to allow the primers to anneal to the complemtary template sequences. then raised back up at an optimal temp to extend or elongate from the 3’ end of the primers. this means that from double standed DNA || it will make two double stranded DNA’s || and ||

The second cycle

the entire steps of heating, cooling, optimal temp is repeated so from two double stranded DNA’s || and || it wil result in four double stranded DNA . it increases the amount of template strands.

In cycle three it will result in 8 double stranded copies.

so theortically 25 cycles yields 2^(25) = 34 million copies.

although in cycle three we see that we have DNA strands that are borered or confined by thhe primer sequences. the primers are the outside borders of the amplified dna.

So the same termpature cycle is repeated many times, and the dna strands that are cycle N serve as template dna’s for cycle N +1, N+2, etc.

The target DNA sequnece double doubles at each sucessive cycle of the reaction. Starting with cycle 3, the reaction begins to produce shorter ddDNA with the two primer seuqneces at either end. Short DNA’s will dominate the reaction.. So not the whole thing is from the origine. In eachh cycle of Pcr the solution is heated to nearly 94 c to denature, although for most enzyme u kill them. So for enzymes like polermarse it is destroyed at >60 celcius which is needed for elongation. so to solve this problem , thermus aquaticus lives in boiling hot springs and it is a bacteria and its Taq dna polymerase doesnt denature at 95C and instead its max activity is at 72. So taq and other thermostable pol are used for PCR. Limitation to pcr is that you already have to know something about your dna sequence of interest , because u need to know how to design complamentary primers.

22D - Sequencing DNA

how do we sequence DNA? we use the dideoxy chain termiantion or sanger sequencing. So it is like pcr where we have a template stand a comlemtary primer and the dna is snythezied 5’ to 3’ . although for this a chemically modified nucleotide is included that contains the sugar dideoxygeribose. these ddNTPS lack a three prime hydroxyl group found in DNA so there is no OH in the sugar. so when we add this to a growing chain of fDNA you cannot have any addition of new nucleotides, no new downstream addition so it terminates chain elongation .

so we have a template, primer, polymerase, and dNTP and ddNTP’s. so we include one ddNTP and dAtp,dCtp, dGtp, dTtp. and we also use dideoxy-GTP. when ddGTP is incorporated into this growing strand it is going to block any further incorportaiton of nucldeotides downstream. We will end up with number of pieces od DNA with their links dictated by where a G was incorporated. you can sepreate these dna links on a polycacrylamide gel that serpate the dna pieces that are only one base pair different in their nucleotide sequence. you see these pieces of dna run at different bands. so in a new synthezied chain, if ddGtp was added in nucleotide 4 then when placing it on a gel it will run the fastes but if it was placed on nucleotide 11 it will be the top band and slowest.

so then you have to perform foru paallele DNA seuqnec ereactions where each raction is going to contain one of the four ddNTP’s ddAtp, ddTtp, ddCtp, ddGtp* and all of the four dNTPs. so gel electrophoresis is going to seperate each band of each dna strnad6 so then we can know where these ddNTps were added and know the sequence is of the snythsized dna strand .

so we see where these markers were placed in each sequence and can read the wole thing

the plasmids used for cloning have large number of restriction enzume site grouped togheter that is called multiple cloning site. ONe dna has been inserted , a primer corresponding to the M13 forward or reverse can be used to sequence thorugh the insert. So now u can sequence the dna of interest u are working with.

onece you have the dna sequenc eyou can consider what protein is encoded by that sequence so you can use the genetic code and predict amino acid sequence and consider what the open reading frames are or the piece of dna that you are studying;

so dna libraries have ben used to seuqnec ethe ntire genome of humans and other organims. this is called genomics . after sequencing a genome there is so much work thaht goes into assmebling that infromation , In the dna library we have all of these plamids containign a little piece of dna, but you dont know how the pieces go together. you dont know which two pieces of dna are next to eachother. u have to assmble millions of sequences in order nd scan and identify open reading grames, look for start and temination sequences to figure out which open reading frame belong to the same gene and compare to know sequences. thisi s bioifnfomraitcs.

Humans have 20,000 protein coding genes, some of these genes mutated cause diseases, knowing the base pair helps, . so genetic screen falls under a classifcation called forward genetics, so when we did our genetic screen we mutagenzied organised and found a mutant animal , found the mutation we mapped the mtuation to a gene and tried to understand what the gene does. if you have genomic informaiton this process can be done in reverse where we start with gene sequence and create a mutation on the gene and then ask what is the impact of that mutation. so you can learn gene fucntion by making a mutation in the gene on purpsoe.

human genome sequnece can be used for cancer biology and detemine seuqnece of fmalignant tumrors and target them.

23 A

how scientist form experiment with rna and protiens

DNA contains genes that encode for RNA and rna for proteins. “gene expression” considering how when and where an RNa or protein is produced from its encoding gene to allow a specific cell type to do its job. So we start with mRNA, we take a tissue grind it up you can isolate all of the mRNA that are expressed in this tissue. You can then convert that mRNA into DNA complemtnary dna that is complentary to the mRNA. this is reverse transcription to make complemtary cDNA, this allows us to infer the nucleotide sqeunce of the mRNA and preict the protein sequence to undestand protein structure and function.

reverse transcription, using rna as a tempplate to make DNA, this is carried about by reverse transcriptase which is a dna polyemrase, it will use rna as its tempalte to make this dna. this reverse transcriptase comes from viruses, so this reverse transcriptase requires a rimer to start . so it adds to the three prime end of the primer.

Eukaryotic MRNA - these will have a poly a tail, we need to design a primer that is going to anneal to the poly a tail. This primer is called oligo DT primer which is a string of T’s, so then mkaing that on single stranded c dna is the product. although sometimes we want to make a double standed dna where we add hexamers which are going to be the primers, 6-mer primers of random sequences. They are going to find comlemtnary sequences along the cDNA. then polymerase dna will synthezie off the three prime end of these random hexamer primers.

what can u do? ligate it into a plasmid and clone it, and then sequence it.

although when u grind up the tissue u have a collection of mRNA’s running thourgh this reactioin so you are convering each mrRNA to cDNA’s so you have a collection of CDNa’s and you can build a cDNA library. sou use a plasmid vector for each cDNA’s and each clone with cDNA library is going to have its own unique cDNA .

so in genomic dna libraries we take a genome and cut it into random piecies so thse enzymes used to cut can cut between a gene, or make it so that a framgnets has pieces of two genes, and has no respect for gene boundaries, we also dont know how these gsequences of fragmetns fit togehter . the library contains all of the dna genome includes sequences that are neighter transcribed or translated.

a Cdna clone comes froma single mRNA and always corresponds to the product of one gene. The library contains squences that have been transcriped into mRNA, this is important since only a fraction of the genome is transcibbed into mRNA. human 1.5% of our dna is transcribed into mRNA. So cDNA comes from mature and process mRRNA so the introns have been removed and the exons re together. know we know what seuqneces are going to be used for protein synthesis or trnalsation. So we can use a cDNA and compare it to genomic dna and then see where the genes will match to the genomic dna and dtermine what is an exon and what is an intron. … if a pre-mRNA undergoes altenrative splicing then a RNA sample can form multiple isoforms of the mature mRNA and these isoforms will show up in a library of cDNA sequences . the protein encodede by that mRNA can be preducted using the genetic code. proteins equence suggest function and structure. so we can look at the open reading froam look for a start and sto pcodon and then see the amino acid sequence. cDNA libaries allow us to understand what gene products are made from gene of interst.

You have a gene of interst and u want to know what the mRNAs tat are encoded from the gene of interst in liver tissue. u can use the cDNA library that you make up from liver to indetifiy gene products that are made form you gene of interst..

So you take the tissue grgind it , collect mRNA’s then collect double stranded cDNA’s and clone them into plasmids to generate the library. plasmids then be put into ecoli or transformed into ecoli and then the bactiera is palted on agar plate. now each bacteiral colony on plate would carry on cDNA and this represent the on mRNA. so now u can use hybridization based stragety , youcan make a labeled dna probe from your gene of interst that will anneal to the cDNA clone in your libarry that has contains inser with complimtary sequencey, you can find those cDNa clones in library thaht have complimentary sequence of your gene of interst, you have ur library plated, use a piece of paper and dab the paper onto eachh of the playes in your library, take it out and with what provides you is a replica of the bacteral colonies, so now u work with the paper called a filter and u allow it to hybridize with ur labeled dna probe, each cdna has it own uniqeue sequence . you doa dtection step and see which it matches to so u known which cdna clone to work with and go back to the plate and pick up that colony.

cDNA - important for medcially important protiens like human insulin which is made from eocli frlorm genes that are cloned into plamids. SO these gens haveintrons an exons but bacteira dont have a way of splicing out the introns from an mRNA. so we use cDNA to clone this cDNa into an expression vector so the introns are gone with the cDNA. so u have the sequence that encode protein and cloned into an expression vector . they have bacteiral promtoor that will allow the cell to transcibethe cDNA and they have ribosome bnidng sites which allow the mRNA to associate with the ribsome and translate.

23 B

grind and find is rinding up a tissue to homogenize . grind it isolate the mRNA then study the mRNAs. or u grind up or homogenize the tisse then the tissue homgenate is spun in order to get rid of lipids and debris and you are left withha. clear rissue extract. this extract cfan then be probed for prescenf of specific rna or proteins,

again tissue, homogenizaiton, chemical sepreation into pools of rna or pteins. want to see if a specififc gene of interestt in in the tissue look for specififc rna using assorted hybrization or u can look for specific proteins using anitbodies .

RNA methods - north blot allows to detect expression of a single gene at a time, you take a tissue grind up the tisse and isoalte the rnas from that tisse and run the rna out on a gel. Ten you wiill seprat ethe rnas on the gel and transfer rnas on aht egel ontoa blot made of nylon or cellulose. to detect if the mRNA is present we tected a specifrfic mRNA the blow would be hydbrized witha complemtary nucelic acid probe that is labeled and here u can see p32.

so in an experiment someone collected 13 diffferent tissus and grounded up the tissue eand isolated the RNA from that tissue. then ran the rna out from the tisue in one lane of a gel. The rna was then transferred to a blot. in each of these blots was hybridizied with a probe for one mRNA. So again rnas were serpeated by size and then blotted onto nictrocellulose and then each blot was probed. Each mRNA is only expressed in some tissues, different mRNAs are expressed differently in different tissues. dark bands - expression of mRNA. nothern blots detects expression of a single gene at a time.

DNA microarray allows to detect expression of thousands od genes one at a time. Microoarray is a solid surface like a glass slide and DNa is dotted onto this glass slide in a pattern. Each dot has a single gene . there is a single standed dna tethered to each of those dots, each dot has many copies of the same single stranded dna sequence tethered to it. THe spotted dnas are corresponding to the sense strand of the genes mRNA.. you you take the tissue, isolate mrnas and syntheeize 1strand cdna’s from the mrna. You include a glorescent label when making the cDNA. the 1st strand c dna is going to in thhe antisense sequence so its going to be complemtary to the mrna strand or to the sense strand. u use a 1st cdna as a probe for the dnas that are tethered onto the microarray. so the cdna will hydrbize to micarray and wher eit finds a sense strand of dna it will be complemtary. A bright dot happens when there is a match. you get a qunaititve picture of how these mRNAs are ebing expressed in the tissue. the intesity of the spot on the microarray is proprortinal to how mnay RNa were present in the tissue extract. the brigh spots indicates there were many mRnas present each of those labeled cDNA is contirbuting to the brightness of the spot. high spots express high expression for that particular mRNA or gene. dim dots suggest no or low expression of that particular mRNA or gene

so by probing a dna mciroarray allows to detect of genes 1000 at a time. the term transcriptone is the entire set of Rnas being expressd by a cell tissue or organism at a giventime .

mRNA seq - allow us to detect thousands of genes at a time. It doesnt require complemtary probe - it identfies the entire RNA eing expressed by cell tissue. based on results u get u can make interpretations of the expression of levels of gene . you can detect unpredicted trasncripts. so for microarray u need to know what ur looking for because that way u know to use a complemtary probe but not in mRNA -seq. So again you take ur tissue isolate mRNA, make a cDNA libarry with adaptor for sequencing primers. these adaptors allow a site for annealing ofa squencing primer. So when these primers are applied you get sequence for each of the RNA’s that was represented in that pool of RNA. And so the n you canuse bioinfromatics toa lign all of the sequences to a reference genoman actually see it.

you grind up tissue, isolat mRNA, make cDNA libaryr with adpters. These adaprter allow sight for primers to anneal and then you get sequenc of RNAs for a pool of rnas. then you use bioinfromatics to align all of the sequences to a reference genom and see how many you get for each exon in the genom, then you can see a reult like this where youre actually seeing hhow many counts in the genom and how many counts crrespond to a given exon.

disatvantage of grind and fine - when you grind any spacial infrfomation is lost. spaital is infromation bc in different tissues not all cells are the same . some genes may be expressed in one cell type but not another. So you lose all of the infromation .

TO get around that we use situ hybridization so we dont homogenize the tissue we are isulating the tissue and keeping it intact. So we keep it intact so the structure is maintained.we fix the tissue witha chemicalthat is going to cross link all of the proteins in the tissue once u fix the tissue, the tissue and the cells in the tissue are dead. So first we syntehize a labeled nucleic acid probe of a known seuqnece, then we hybrize probe to mRNA ina population of fixed an permabilized cells. then wash out unhrbized probe and visual the labeled cells.

the cells are fixed and glue down, so the cell is permabilized and the labeld probe will enter the tisse and look for complementary mrna in the cell and anneal and then u wash out the unbrized probe and see where the labeled probe appears in the cell.

considering for probed in situ - in order to hybrize with the targeted m RNA in situ the probe must be complentary, because the mRNA itself represense thhe sense strand the complentary probe must be the antisence, in situ probes can be dna or rna, probens can be labeled with fluorescent tages allowing FISH. flourscense in situ hydrbization.

23C ahhhhhhhhhh

so we are going to detect specific protein can be detected with antibodies - this can be done through westner blots. so you take the tissue homogenize it then purify the tissue extract with the proteins then run those proteins out on a gel to sperate by size. then proteins are trasnfered onto a blot and blot probed with antibodies again the protein of interst, the antibodies will bind to the protein of interst, and bound antibody detected with labeld probe. again so thhe positition of protein remain the same on blot. you take the blot incubate the blot with antibody against the protein of interst and if the protein is present the antibody is going to bind to the protein then it will light up that particular band.

proteins were serpated by size and transfered to nitrcellulose, each blot was probed witha specific antibody to detect a specific protein, spt protein is expressed at a relavitly constant level over the six sages, sr a prteoin is expressed in a pulse witha peak expression at stage four. so the protein extracts were prepared from embroys at six developmental stages. “

so in a western blot u lose infromation in the tissue because of the grinding. u can get around this problem for looking for proteins by using immunohistochemistry. so you work with a fixed intact tissue, the tissues architecture is maintained. you introduce a primary antibotidy against the protein that you are intersted in, if the protein is present the antibody will bind to a protein, to detect where the primary antibody was found you use a secondary antibody. this antibody will reconize an antibody,. the secondary antibody is linked to an enzyme , so you add a ragent and then the reagen will interact with the enzyme and create a color . another way is instea do using histochem u can use immunoflourescense, where the primary antibody is labeled witha fluroscent tage, or the secondary antibody has the tag.

24 a

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