Genomics - All the experiment techniques

Quantitative PCR/Regular PCR

*to determine what kind of RNA is present in a sample extract RNA --> cDNA --> carry out PCR reaction -use reverse transcriptase to convert RNA to a DNA sequence and makes it more stable and easier to use (convert to cDNA) -what kind of RNA is present in a sample -quantitative: how much RNA did we start with that we extracted from a sample -seeing if RNA is present at all, in this case would just look for a band on a gel -ex. quantitative PCR used to determine levels of HMOX1 RNA in the patient's liver cells as compared to levels in liver cells of patients who do not have the disease.

Mutant Mapping - Identifying Mutations

--> 2 approaches to understanding underlying mechanisms 1. Reverse genetics - start with a gene -get rid of it (remove it and see that does to the phenotype of the organism) -or make too much of it --> see the effect on the phenotype => both get you to phenotype (figuring out function of gene) 2. forward genetics -start with a phenotype and you try to find the underlying genetic cause (specifically what gene is mutated that causes this phenotype)

CSI (crime!)

-DNA from the crime scene -DNA from a suspect --> asking: Are these the same? -PCR up certain fragments of DNA where they know that there is restriction polymorphism & cut with the enzyme -if the same, should give the same pattern on PCR -PCR for SSLPs --> patterns should be the same (same SSLPs) -SNP analysis --> are the same SNPs present in the two samples?

"Zoo" blot

-DNA sequence of interest --> radioactive probe -tells you if DNA sequence you're interested in is also present in other species (more old-fashioned lab way)

DNA sequencing - Sanger/Chain Termination sequencing

-Developed by Frederick Sanger --determining the peptide sequence of insulin --developing a strategy to sequence DNA -DNA replication by DNA polymerase is fast -spike in chemically altered bases (A, G, T, C) into the reaction to stop it

Genome Editing with CRISPR-Cas9

-Genomic characterization of potential unintended genome editing with CRISPR/Cas9

Microarray analysis

-able to detect thousands of transcripts in one experiment (unlike other techniques we only are detecting/looking for one type of RNA molecule) -era of transcriptomics --> can sample and see all transcriptome changes/changes in gene expression that occur when changing environment of cells, ex. drug (instead of one by one)

Homology

-all living organisms descend from a common ancestor -exons: are highly conserved -introns or antigenic sequences - are less conserved, more variable -showing that chimps mouse, and yeast and namatode have the ORF shows it's highly conserved, meaning it's an important gene!

SOLID sequencing

-another NGS technique -relies on hybridization rather than DNA synthesis -rather than taking new DNA strand, taking 5-mer oligonucleotides and hybridizing them to DNA sequence of interest, and then can figure out which 5-mer nucleotides/sequences annealed to our target DNA and from there figure out what the sequence of the DNA is -1024 5-mer nucleotides 1. 5-mer oligonucleotides 2. first oligonucleotide hybridizes and is ligated to primer 3. Second oligonucleotide hybridizes and is ligated -very accurate, but cannot do it for long stretches of reads (also ways to read DNA without replicating it)

homology search

-at its core, homology search works by alignment -query sequence (Gene X) (sequence put into BLAST) is compared to every sequence in our database -the number of positions at which you have the same nucleotide or amino acid is located -can compare both the DNA sequence or the protein sequence - often the protein sequence is more powerful -this gets translated into a score => use BLAST to do this homology search

Continue biolormatic approach

-carry out a homology search --> locate genes by comparing the DNA sequence of interest to all other DNA sequence in a database -database is fast and for free

knockout

-commonly used technique to inactivate a gene => completely remove the gene sequence from a cell or organism -gene inactivation by homologous recombination -The vector DNA carries two segments of DNA that match the ends of the gene to be inactivated. -These segments recombine with the chromosomal copy of the target gene. => homogenous recombination -As a result, the target gene in the original chromosomal DNA becomes disrupted. -if also want to see how gene expression changed with knockout, perform RNA seq.!

SNP mapping

-crosses -to carry out SNP mapping, have to introduce some diversity into the kinds of SNPS that are present in the organism -looking for where mutation is lying in the genome *point at which mutation exists, only going to have N2 SNPs, no recombination because only selecting for the mutant phenotype --> *where there are no Hawaiian SNP, means no recombination, suggesting that is where the mutation is located because mutation has no recombination

Genome sequencing graphs for C. elegans experiment

-did whole genome sequencing, and figure out which part of the genome there is missing Hawaiian SNPs -SNP mapping can direct us to what part of the genome is responsible for the mutant phenotype we see => now we can zoom into this and the genome browser of C. elegans and see genes present in this interval -so both whole genome sequencing allows us to figure out where are all the Hawaiian SNPS located, as well as are there any mutations in any of the genes in that particular interval

reverse genetics

-discovering gene function from a genetic sequence -start with a gene --> want to identify its function

SSLP

-done with PCR --design primers that span the repeat area --amplify that area and run it on a gel to see if there's a bigger band (you have more repeat - DNA fragment is larger), or smaller band (you have less repeats - DNA fragment is smaller) -capillary electrophoresis: separate out DNA sequences of different length

gel electrophoresis

-electrophoresis: movement of charged molecules in an electric field-DNA: negatively charged + linear-agarose or polyacrylamide: network of pores through which DNA fragments passseparates DNA fragments by size

Next generation sequencing

-entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences. 1. cut with restriction enzymes 2. put on DNA chips --> oligonucleotides -oligonucleotides have complementary sequences to the adapters 3. after attached DNA fragment using the adapter sequence, you carry out PCR reaction on the entire chip: to amplify -whole chip will go through PCR process -duplicating DNA fragment so that entire spot on ship has particular fragment (way fo amplifying up DNA fragment to get more accurate sequencing read) -fragments of DNA are single-stranded -each spot has a different DNA fragment 4. sequence all at once on chip

Hierarchal shotgun sequencing

-first prepare a clone library of large DNA fragments 300 kb (1.6-2kb) --> clone into BACs -clone contig - overlapping, large fragments of DNA -"chromosome walking" -start with larger clones -sequencing large piece of puzzler rather than al the smaller pieces -better in situations with receptive sequences (know where repetitive sequences are)

2 approaches to genome mapping

-genetic mapping/linkage analysis: relies on the observation of inheritance patterns during enticing crosses -physical mapping: using molecule biology techniques to directly identify features in the genome

How CRISPR works

-guide RNA - 20 base pairs long --> design it to be complementary to your DNA sequence of interest -two components: -guide RNA sequence is upstream of NGG or NAG at the end (these need to be down stream of where your guide RNA will bind) -Cas9 endonuclease - follow the guide RNA and create double-stranded breaks at the site where the guide RNA is steps: 1. Guide RNA binds to target sequence 2. Cas9 enzyme binds to guide RNA 3. Cas9 enzyme cuts both strands of DNA 4. The cut is repaired introducing mutation --> introduce other DNA sequences --> possibilities for editing site -if we target our Cas9 endonuclease, for example the first exon of the gene we are trying to inactivate, can easily introduce a mutation, which then results in the inactivation of that gene and cannot produce a functional protein -in additional, once created the double stranded break can also supply cells with vector DNA that can "repair" this sequence but can introduce new sequences into our DNA - just like with homologs recombination can introduce other DNA sequences once it has been opened up -opportunities for editing a particular site in the genome using CRISPR Cas9 system -now we know we can inactivate our gene either from homologous recombination or more modern way with CRISPER Cas9 -would still happen in ES cells

Hierarchal clustering and heat map

-hierarchical clustering - comparing expression levels of every gene pair of genes in a transcriptome and grouping based on similarity of expression pattern --should group together all the genes expressed in sample 1 but not in sample 2 and vice versa and all the gene expressed in both samples -can put together dendrogram after hierarchical clustering- way of visualizing genes with related expression profiles, which are clustered together -way to visualize microarray data - heat map: --green - low expression --red - high expression -genes at time points -can see that overtime (takes time for machinery to do its job and for transcription to occur, so takes longer to see results), certain genes are up-regulated and some down-regulated because of change in sample --> can see how that happens with this technique

Now that we have tools that we can tag different proteins - able to see distinct structures in the nucleus

-images of living nuclei obtained after labeling of proteins with GFP: -blue fluorescent light spot in image - nucleus -pink/red spots - canal bodies --> site where small nuclear and small nucleolar RNAs are made

Molecular cloning

-is a set of techniques used to insert recombinant DNA from a prokaryotic or eukaryotic source into a replicating vehicle such as plasmids or viral vectors.-uses enzymes that are naturally occurring that are used by cells for replication, repair, recombination, defense

knockout or transgenic mice

-mouse is the best model for humans -have to carry out deletion process very early on -carry out process of recombining gene of interest and putting in deletion cassette in embryonic stem cells --> ES cell (right after fertilization) -totipotent: not committed to a particular developmental pathway and can give rise to many different cell types --> cells that are going to become the germline/gametes

ATAC seq

-number of assays to determine which regions of the genome are open and therefore actively transcribed -another way to do this is looking at sensitivity to DNase 1 activity (talked about earlier) - open areas of the chromatin would be sensitive to DNase activity

Northern Blot

-old-fashioned way used when you want to detect if a particular RNA molecule is present in sample

FISH

-one way to physically map gene sequences onto your genome of interest is through a technique called FISH = fluorescent in situ hybridization --can isolate chromosomes from cells, fix them on microscope slide, denature them, and then add a probe which is a short DNA sequence that is complimentary to DNA sequence of interest -creating DNA probe complimentary to gene sequence (part of it) -probe is labeled usually with fluorescence dye -so you can follow onto which chromosome probe is binding to and where along the length of the chromosome it is binding to

shot gun sequencing

-pieces from throughout the genome have been randomly sequenced and computers order the fragments -small pieces of genome are sequences, and the sequences are then assembled using a computer. and gaps between the pieces then have to be found and sequenced -starting with genomic DNA and shattering into short DNA fragments -big computational component --> putting small fragments all back together into whole genome -still works, but hierarchal sequencing is slightly better (like in Human Genome Project) -dont' know exactly where repetitive sequences are (don't know in which part or chromosome it's in), reads are very short

RFLPs

-polymorphism means it exists in different forms -differences in homologous DNA sequences that are reflected in different lengths of restriction fragments produced when the DNA is cut up with restriction enzymes -different DNA sequences have different restriction sites (different amount of restriction sites) = variations in sites in DNA can be cut by different restriction enzymes -variations that arise naturally in evolution -technique used for this is Southern Blotting -take genomic DNA sequence and cut it with restriction enzyme -because there are many sites for restriction enzyme --> you get column full of DNA fragments -transfer DNA in your gel on a nylon membrane -probe nylon membrane with DNA probe (incubate together) --> DNA probe is a complementary sequence to your sequence of interest, and is also radioactive -going to bind to the DNA sequence that is complementary to DNA probe -can see where DNA probe bounded to nylon membrane --> shows DNA sequence of interest

Uses of PCR

-sensitive test: starting with nanograms of DNA and through PCR --> no get micrograms of DNA -making DNA detectable uses: -screen DNA for mutations -genetic profiling -recovering DNA sequences from ancient humans -COVID-19 test

Single-cell RNA-seq (ScRNA-seq)

-sequencing RNA in one individual cell, one cell at a time -can see clusters of gene expression similarities that show different cell types

ChIP-seq

-technique to figure out where in the genome TF bind to and get determine this inside of a cell

DNA regulatory sequences - Turning genes on and off

-usually exist upstream of gene of interest that regulate turning gene on or off -upstream of gene of interest are regulatory sequences --> -regulatory sequence has enhancer + promoter that is upstream of gene X (gene of interest) -promoter is right upstream where RNA polymerase would bind -enhancer are further upstream and is where transcription factors bind to and affect if gene will be expressed -to over-express our gene of interest (gene X) to figure out the function of that gene--> regulatory sequence is always on -would use cloning tools in recombination DNA lecture to create DNA construct -this creates a transgenic mouse --> gene of interested is over-expressed and regulatory sequence is always

RACE

-when identified candidate ORF, but want to know exactly where the start site is = rapid amplification of cDNA ends -start with RNA and make cDNA with reverse transcriptase -denature strands and anneal primer -can start to synthesize new DNA strand -have double stranded DNA that can be used as a regular template for PCR reaction --*sequence your PCR reaction --> will now include the 5' end of your gene of interest -repeat process for primer to anneal on 3' end of your gene -*ultimately, you can characterize the beginning and the end of your gene -shows what is the 5' end of our gene and repeat to find 3' end of our gene (-primer that anneals -reverse transcriptase -anchor primer -synthesize new DNA strand) -*sequence your PCR reaction --> will now include the 5' end of your gene of interest -now can characterize and map out your gene -specifically characterize the beginning and end of your gene

454 sequencing or pyrosequencing

-when nucleotide is added - can be detected with camera -uses chemiluminescence -reads can be longer (1000 base pairs)

Mapping the Human Genome

-where are all the genes located? -not trivial to ask, what part of the genome is encoding for a protein, versus what are the non-coding parts of the genome -if we want to identify DNA sequences that encode for protein, we start with mRNA. -- take mRNA from a cell and want to turn RNA sequence back into the DNA. --can then use this DNA sequence to probe our genome of interest and see where is this DNA sequence that must be encoding for a protein located => this is the process of creating cDNA: RNA --> cDNA (coding DNA) -take mRNA isolated from cells and we know that mRNA's have polyA tail -can create oligo(dT) primer which is a sequence of T that will hybridize with Poly A tail -this is our primer used to create DNA strand using RNA strand as a template -special enzyme for this: reverse transcriptase - polymerase that used RNA as a template to make DNA -get double stranded DNA sequence that was created using mRNA as a template => this is our cDNA *cDNA not going to be the same as the genomic DNA -cDNA would only include exons (genomic DNA includes introns) -what you can still do is if you take cDNA sequence form reverse transcription, you can use that as a probe such as in FISH experiment to figure out where gene encoded by cDNA is located in the genome -it will still find genetic DNA locus and hybridize with that locus and can figure out where this gene is located in the genome

Which alleles and how many underlie a phenotype?

-whole genome sequencing (if looking at it in a disease, can compare whole genome sequencing of control patients (no disease) to patients with the disease -whole exam sequencing -SNP analysis

Protein binding microarray

-will tell you exactly the DNA sequence that is being bound by the TF

Typically MS-based proteomics analyze peptides, NOT proteins

1. extract proteins from sample (cells) --> 5,000 to 15,000 proteins 2. digest with a protease (typically trypsin) --> 100,000 - 1,000,000 peptides 3. analyse sample on mass spectrometer analyzing sample on mass spectrometer: -The mass spectrometer will sequence peptides -computational methods elucidate proteins from peptides

Database Use - steps

1. identify a new gene 2. translate into protein 3. perform BLAST search - will tell us orthologs of protein -can also look for conserved domains with Prosite website 4. from this can figure out what protein family is our protein of interest in -transcription factor?, eynzme?, etc. next step: want to inactivate the gene and look for a phenotype -down-regulate or completely remove the gene from the genome (next flashcards talk about strategies to do this)

Summary of Understanding Gene Function

1. inactivate the gene --> look for a phenotype -inactivate either with CRISPR Cas9, knockout with homologous recombination , or RNAi. -ways to turn gene off with regulatory sequences 2. overexpress the gene product --> look for a phenotype -make transgenic mice: overexpress gene product -ways to turn gene on with regulatory sequences 3. determine where and when the gene is expressed -PCR amplification -GFP -immunofluroescence

How to detect RNA

1. northern blot 2. quantitative PCR/regular PCR 3. fluorescent in situ hybridization (FISH) 4. microarray analysis 5. RNA sequencing *detecting RNA can therefore tell you expression level of a gene

analyzing gene we have not studied before

1. predicted the existence of a gene in the human genome that has not been studied before 2. find/isolate mRNA molecules/sequence from an individual that correspond to our predicted gene (gives strong evidence that this gene X is expressed in cells) 3. use DNA sequence to predict the amino sequence of our gene of interest => continue bioinformatic approach by carrying out homology search

To determine the lifetime of a mRNA molecule: perform pulse-chase experiment

1. provide a "tagged" substrate that will get incorporated into our RNA molecules ie. radioactive 4-thiouracil --> now we have labeled RNA 2. follow "tagged" RNA molecules until they disappear (meaning degraded by cell) and time this -in bacteria, mRNAs are degraded by the degradasome: multi protein structure which removes nucleotides, sequentially from the 3' end -in eukaryotes - structure is called exosome Other degradation processes: -Synthesis of miRNA from a holdback RNA precursor -funtional miRNA incoporated in RISC complex (RNA induced silencing complex) -within complex, endonuclease called Argonante - endonuclease that cleaves RNA -targeted by Argonante -Binding site for miRNAs are often in the 3'-untranslated region of the target mRNA.

Recap of experiment

1. starting with wild type animals expressing a fluorescent marker in neurons so we can visualize the morphology of neurons 2. took these animals and mutagenized them with the chemical EMS that introduces random mutations into the genome of these animals that allowed us to isolate a mutant that has striking phenotype of having very short dendrites 3. analyzed mutant by performing several crosses and determined it was a recessive allele - needed two copies of mutant allele to see the phenotype 4. then performed SNP mapping coupled with whole genome sequencing to identify an interval in the genome that is carrying this mutation that is causing this phenotype 5. in that interval identified 10 candidate genes - can go through those genes one by one determine from the whole genome sequencing if any of these genes have any mutations in that them that was introduced by EMS 6. then rescue the expression of that gene in our mutants - expression of the gene that will rescue the phenotype is our best candidate --> turned out that gene dyf-7 causes the abnormal dendrite: -Mutation in the gene dyf-7 causes abnormal dendrite morphology -point mutations in DYF-7 have varying effects on dendrite length

Recombinant DNA

= DNA sequences that have been formed artificially (by people) by combination from different organisms-involves: molecular cloning-enzymes used in recombinant DNA: enzymes that are naturally occurring that are used by cells for replication, repair, recombination, defense

Genetic Markers

= a gene or short sequence of DNA used to identify a chromosome or to locate other genes on a genetic map. other features in genome that are genetic markers: 1. restriction fragment length polymorphisms (RFLPs) 2. simple sequence length polymorphisms (SSLPS) 3. single nucleotide polymorphisms (SNPs)

Polymerase Chain Reaction (PCR)

= amplify specific DNA sequences many, many times-uses the principles of DNA replication-in vitro = in a test tube- in vivo = inside the cell Ex. Start with piece of DNA that is 5kb in length- want to amplify part of it-would design your primers for a certain region: forward + reverse primer -as you are carrying out the 3-step process 30-40 times, the short product (the product of the DNA sequence between the two primers) will be amplified over and over again-to the point where you can get 130 million copies of this DNA sequence-can start with nanograms of template DNA --> micrograms of amplified DNA (can use in recombinant DNA technology)-how you know your reaction worked: if amplified region should measure to 2kb, can run PCR product on a gel and see if you have a band that corresponds to 2kb

ORF scanning

= looking for ORF is a way to predict DNA sequences that encode for protein -Effective way of locating most, but not all of the genes in the DNA sequence -Analysis of bacterial sequences is simplified = don't have non-coding introns looking for: -exon-intron boundaries: because genome includes introns so hard to tell which ORFs are just in exons, that are actually expressed in mRNA -ways to predict splicing site: -*consensus sequence: sequence that shows the most frequent nucleotide at each position -codon bias (some codons used more frequently than others) --for example, leucine is encoded by 6 codons, but most frequently CTG -upstream regulating sequences: --CPG island located upstream of gene and ORF --CPG island has high GC content (lot of G and C nucleotides) --another way to scan for ORF, which is a sign for an ORF that gives a gene coding for a protein in addition to scanning for ORFs, another way to look for mRNA sequences that encode for protein: -can also scan genomic sequences for non-coding RNA -tRNA is an example of non-coding RNA

How can we detect TF binding to DNA?

=> Gel retardation/mobility assay -or DNase I footprinting (more accurate assay)

Restriction mapping

=> another technique used to physically map genomes -taking DNA sequence of interest and cut it with different restriction enzymes -take a look at figure 3.28: show example of how when you have a short DNA fragment you can cut it with different restriction enzymes and can piece together a map for where those restrictions sites must be located based on the fragments you get -works well for a short sequence of DNA or small plasmid -falls apart if you're trying to analyze an entire genome --> so many restriction sites that overlap (not effective for entire genome) -optical mapping fixes this problem

association testing

=> association testing to determine if one allele is more represented than others: -calculate the odds ratio

nucleosome remodeling

=> changes in the positioning of nucleosomes so that DNA-binding proteins can access their sites -a second type of chromatin modification that can influence genome expression -ways to do this: -remodeling: loosening up how tightly the DNA is wound around a histone -sliding: can slide nucleosome over for part of DNA to be available for binding RNA polymerase/transcription -transfer: can transfer nucleosome from one DNA strand to another DNA strand; would also result in DNA area being open for gene expression -the way we can read this out: DNase I sensitivity assay --take DNase 1 which is an enzyme that randomly cuts DNA --when DNA is wound around histone and forms nucleosome, that DNA is protected from being cut by enzymes --areas in the genome where you could cut with this enzyme suggests that this is an open part of chromatin --> these areas/open parts are all located upstream of the gene cluster --> suggests this is the parts of the genome bound by transcription factors and bring in RNA polymerase to transcribe the genes -if we find these hypersensitive sites upstream of genes, suggests that these areas must be opened up and are important for activating transcription

Bioinformatics

=> computational analysis of genome sequence -open reading frame (ORF): DNA sequences that likely encode for protein -bacterial genes are 300-350 codons -human genes are 2450 codons

Genetic screen

=> take animals that carry these markers so we can easily visualize neurons, and mutagenize them that causes mutation in their progeny and screen the progeny and try to find a mutant animal in which the neurons did not form properly so that there is a clear observable defect in the neurons

Radiation hybrid

=> technique for mapping mammalian chromosomes -X-rays --> cause fragmentation -form hybrid chromosomes (ex. hamster plus human) -markers can test positive, meaning markers are close together -ex. for micro satellite markers -can use cDNA as markers for where genes are located in comparison to other markers

Another way you can visualize when and where you gene of interest is expressed: immunofluorescence

=> using antibody that is specific to your protein of interest-antibody carries fluorescent tag so you can visualize it-use this antibody and it binds/detects where gene of interest is expressed by binding to protein product

Clone library

A collection of cloned DNA fragments; also known as a gene library. -can be used to physically map DNA -previously, we talked about making plasmids: can take gene of interest and clone it into a plasmid and easily propagate that into bacteria cells -another way we can use this technique for is to isolate a specific chromosome (ex. human chromosome 2), cut it up with restriction enzymes that will create DNA fragments of varying sizes that will span the entire chromosome, and ligate that into plasmids. -now each plasmid will carry a unique DNA fragment that is coming from this chromosome -can use these plasmids to transform them into bacterial cells that will eventually form colony -each colony will have a plasmid that will contain a unique DNA fragment => how we create a clone library -clone library = a collection of vectors carrying DNA fragments --usually large DNA fragments: several kilobases -probably will use a BAC (bacterial artificial chromosome) vector (for large DNA) *can design PCR reaction to detect our markers of interest -go through all our clones and see if they are carrying the DNA sequence of interest that has a marker -trying to figure out if markers are close together -if markers are physically close together --> many clones will be positive for both markers -alternatively, if markers are physically far apart on chromosomes, when we are screening through our clones in our clone library, we rarely find that a particular clone tests positive for having both markers (suggests two markers we are looking at are not that close to each other physically on the chromosome)

Southern Blotting

A hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA. -method used in molecular biology for detection of a specific DNA sequence in DNA samples -use DNA probe - complementary sequence to your sequence of interest --radioactive

RNA sequencing

A method used to determine the transcribed regions of a genome within some specific cell population, tissue sample, or organism. *determining expression level! technique used to detect transcriptome of a cell/sample -look at what are the RNAs present in the sample -overtaken most strategies, more commonly used -takes advantage of Next-Generation Sequencing (NGS) to determine the transcriptome of a sample

Fluorescent in situ hybridization (FISH)

DNA probe that is fluorescently labeled --> hybridize it to RNA at the level of cells or tissue or an entire embryo -trying to determine where is our RNA species expressed ex. image of section of brain: -red - mRNA for Gene X -green - mRNA for Gene Z

When and where is Gene X expressed - using GFP

Gene X reg. sequence = upstream -in order to understand when Gene X is expressed we can take regulatory sequences and remove our gene and instead hook it up to expression we can easily follow --> protein called GFP -wherever we see GFP protein, we would know our Gene X (gene of interest) would be expressed there -GFP would be downstream from regulatory sequences *GFP => green fluorescent protein -glows brightly when shine wavelengths of light on it -can tag protein with GFP as well -comes from jellyfish -different regulatory sequences light up differently with GFP --> can use GFP to mark specific cells ex. regulatory sequence upstream of bone-specific gene --anywhere developing into bone lights up --hooking up regulatory sequences to GFP

Gene inactivation with RNAi

RNAi - RNA interference = > one way to inactivate a gene --> create double stranded RNA that is complementary to our mRNA sequence of interest --> Dicer nuclease cuts the double-stranded RNA, and single-stranded siRNAs attach to the mRNA and mRNA is cut by Argonaute 1. The double stranded RNA molecule is broken down by the Dicer ribonuclease into short interfering RNAs (siRNAs) of length 21-25 bp. 2. One strand of each siRNA base-pairs to a target mRNA, which is then cleaved by an endoribonucelase called the Argonaute protein => will either prevent the translation of this mRNA or cause its degradation => results in down-regulation of expression of our gene of interest in whatever cell this RNA molecule has been introduced to -RNAi has been used extensively in C. elegans (very easy to use organism) --worms eat the RNAi-bearing bacteria --> results in gene knockdown throughout the organism --> only organism where we know the RNAi can travel within organism Targeted Gene Inactivation Using RNAi: -The basic concept is that the antisense sequence hybridizes with the normal mRNA in the cell and either blocks translation of the mRNA or increases the degradation of the host mRNA. *cons with this process!

Test Cross

When we don't know the genotype of parent I -In what order are genes A, B, and C? -result of test cross answers this -can figure out order to genes from the test cross based on the frequency of recombination (tells us placement of genes on chromosomes - gene mapping!) -through test cross, can determine the order of genes A, B, and C along the chromosome based on the frequency of recombination based on the test cross

GWAS (genome wide association study)

a method for identifying chromosomal regions containing genes whose variation influences a disease phenotype by using millions of genetic markers scored for hundreds or thousands of individuals who have the disease and who do not have the disesae -collected genomic DNA from research participants (blood sample) -population of 1000s --> case (1000) vs. control (1000) -visualize with Manhattan plot

Illumina sequencing

allows you to get billions of bases on something the size of a microscope slide -another form of NGS: => use the beads talked about! -DNA sequencing reads are short about 300 bp in length -much faster: generating a lot more DNA sequence --2,000 mb of DNA sequences in one go/reaction --whole Human Genome Project was 23,000 mb

How many genes are there? How are genes arranged in the genome?

approaches: 1. bioinformatics 2. homology 3. transcript mapping

where our gene of interest has just 1 point mutation

create cassette that's going to be used to replace the genomic sequence that's present in wild-type animals: 1. design primers that contain mutation 2. anneal mutated strand 3. final PCR cycle => end up with larger DNA fragment that has point-mutation (can use this as targeting construct) => this is how you determine in which DNA sequence a mutation lies --> 1. PCR amplify 2. send for sequencing You have access to the patient's genomic DNA. How would you determine in which DNA sequence (exons, promoter, or enhancer) the mutation lies?: PCR amplify the exons, promoter, and enhancer sequences off of the patient's genomic DNA and sequence them. Compare these results to the reference human genome. OR Perform whole genome sequencing of the patient's genome and compare their HMOX1 promoter, enhancer, and exon sequences to the reference human genome.

What gene is disrupted that causes this phenotype? - Isolating a mutant - first determining if the mutant allele causing this phenotype is dominant or recessive

cross mutant to wild type male: -if the heterozygous animal looks like mutant (meaning it has short dendrites) = dominant -if the heterozygous animal looks like wild type = recessive -and do linkage analysis through crosses

gene association

figure out whether or not when one or more genotypes within a population co-occur with a phenotype more often than would be expected by chance -used to figure out what are all the genes that determine height of example and which ones are the ones you have to have in combination of

Figuring out function of TF

get rid of TF in model organism, to determine what happens in the neural crest cells in the absence of FOXD3 --> tells us its function -1. create a "knockout" -2. perform RNA-seq -perform RNA-seq on wild-type neural crest cells and FOXD3 mutant neural crest cells --> we know FOXD3 is a transcription factor that regulates down stream gene expression so might be interested in understanding what are the genes it is normally turning on or off in wild-type (those genes should be disregulated in FOXD3 mutants and should be able to pick that up by RNA-seq)

Transcript mapping

if our ORF encodes for a protein, we should be able to isolate mRNA -to examine RNA, we have to run a *Northern Blot: detect presence of RNA -then transfer RNA on nylon membrane and take DNA sequence of interest and turn into radioactive probe (like we did with Southern Blot) => in other words DNA sequence of interest and turn it into radioactive probe -wherever the DNA probe finds complementary RNA, it will bind there, the gel will show band at that position --complementary strand, there will be a band -if we see the band, shows good evidence that the ORF sequence makes the RNA to be transcribed to eventually make a protein

How can we recognize when the odds ratio is significantly different?

p-value

To understand if chromosomes have set territories in nucleus/location (chromosome painting:)

perform technique called chromosome painting: -after division, the chromosomes become less compact and cannot be distinguished as individual structures unless special technique like chromosome painting is used -a variation on FISH (before used to know where in the genome a particular gene sequence is located, but now know this) -make probes for genes on different chromosomes:

probe

short DNA sequence that is complementary to DNA sequence of interest

SNPs

techniques for detecting SNPs: (A) Hybridization with an oligonucleotide with a terminal mismatch (B) Oligonucleotide ligation assay (C) The ARMS test (can run products on a gel) -specific SNP analysis process (can't use Southern Blot or PCR) -use hybridization -look at 300,000 SNPs from all across the genome -DNA chip is hybridized to labeled DNA: wherever there is a positive signal, means you have that variant (that SNP)

Where and when is a TF expressed in species?

two ways to do this: -tag TF with a fluorescent protein so you can make transgenic animals and then image those animals and look to see where there is expression of the fluorescent protein, like GFP for example (most likely the preferred method) -another method: perform a single-cell RNA-seq. experiment - take entire organism and sequence all the cells that are found there one by one and see which cells express TF (expensive way)