Biotech Ch 8

Highly Repetitive Sequences

- Eukaryote genomes have two types of highly repetitive sequences that do not code for proteins - Number of copies varies among individuals which allows then to act as molecular markers Minisatellites: - aka VNTR - 15-100 bp long, repeated 20-50 times Microsatellites: 2-6 nucleotides, and are present in tandem arrays of five to about 30 copies

ribosomal RNA genes

- Example of moderately repetitive sequence - 18S, 5.8S, 28S are transcribed as a single precursor molecule - Humans have 280 copies of the sequence on five different chromosomes - S = svedberg units

long interspersed elements (LINEs)

- Long, repetitive sequences found interspersed in the genomes of higher organisms - LINE sequences are transcribed into RNA and translated into proteins. One of these proteins reassociates with the mRNA. TYPES - LINE-1 family < - LINE-2 family < - Retrovirus-like elements (500-1300 bp) ∼250,000 copies < - DNA transposons (variable; average ∼250 bp) ∼200,000 copies

Unique Genomic Elements

- Protein-encoding genes (includes regulatory regions, exons, and introns) - Noncoding RNA Genes (snRNA, telomerase RNA, Xist RNA) - pseudogenes - Nonrepetitive intragenic noncoding DNA < - Interspersed repetitive DNA <

Short Interspersed Elements (SINEs)

- Repetitive sequences found in the genomes of higher organisms - range in length from 130 - 300 bp; 15 percent of human DNA - Usually contain only an internal promoter for RNA polymerase III - ie The 300-bp Alu sequence which is present in a million copies

histone genes

- The histones gene family consists of major genes (H1, H2A, H2B, H3, and H4) - In Drosophila, these 5 genes occur in a cluster of about 5000-6000 bp, and each cluster is tandemly repeated between 100 to 1000 times. - In higher eukaryotes, a cluster of histones genes exists at only 10-40 copies per genome

Transposons (jumping genes)

- Type of LINE Element that can move from place to place in the genome - make up 40 percent of human genome - only 3 - 10 percent in other sequenced eukaryotes < - not tandemly repeated but exist as isolated elements that may be present in thousands of copies per genome

Components (sequences) of Mammalian Genome

- Unique Elements - Moderately Repetitive Sequences - Short Interspersed Elements - Long Interspersed Elements - Highly Repetitive Elements

DNA microarray +

-A method to detect and measure the expression of thousands of genes at one time. - contain thousands of different unique functional DNA sequences bound to a solid support (chip) - there is a probe on the slide and the target sample is in solution - the microarray includes sequences corresponding to each gene in the organism. - When fluorescently labeled RNA is incubated with the microarray, complementary sequences hybridize. The level of fluorescence corresponds to the amount of RNA that is bound to the DNA microarray. https://www.youtube.com/watch?v=VNsThMNjKhM&ab_channel=Proneural

RNA Seq clinical applications <

-In cancer research, RNA-Seq can reveal if the fused genes are expressed into mRNA and estimate the relative abundance of the fusion product. -identify expressed single nucleotide polymorphisms (SNPs). This type of information can identify the genes responsible for a particular disease by comparing the expression of SNPs from affected individuals and their healthy family members. -identify post-translational editing of mRNA that is not evident from looking at just the DNA sequence. This can suggest a new function for a gene.

major roles of epigenetic regulation in mammals

1-Genome integrity: most genomes consists of assorted mobile elements. Uncontrolled replication or movement of those still active could cause major damage to the genome. These elements are covered by methylation and histone modifications. 2- X chromosome inactivation: females one X chromosome is inactivated by methylation 3- parental imprinting:in a few genes only one of the pair of alleles is expressed, this depends on whether the allele came from the mother or the father, the non expressed allele from the other parent is silenced by methylation and histones. 4-Development and differentiation: all cells share the genome but have different biological roles, once cells have reached a final specialized form, they often rely on epigenetic modifications to ensure that all their descendants are of the same type.

RNA seq steps +

1-ISOLATE mRNA BY BINDING TO POLY(T) BEADS 2-CONVERT TO cDNA 3-SEQUENCE WITH NEXTGENERATION TECHNIQUE 4-These sequences are then aligned with the genome of the organism. 5-The relative copy number of each cDNA sequence is an indication of gene expression levels.

Photolithography Microarray fabrication

1.) A glass slide (quartz wafer) is first covered with a spacer that ends in a photosensitive blocking group (photolabile) that can be removed by light 2.) A mask that blocks light controls which nucleotide gets added on next by controlling which blocking groups get removed. A mask is designed specifically for the round and nucleotide that is added next. So if you want a series of features with probes that are 3 bp long, you need to have 12 masks. (since they are specific to the round and base pair in question.) 5.) Each nucleotide has its 5′-OH blocking group that comes off when activated with light. 6.) Once the blocking groups are removed, a solution of a nucleotide with blocking groups are added in which grow the oligonucleotide. There will be 4 solutions, one for each nucleotide. Start at 3:26 https://www.youtube.com/watch?v=DjcxE1tdB8c&ab_channel=SupportCenterforMicrosystemsEducation

Bisulfite Sequencing Steps

1.) Scientists get 2 sets of a DNA sequence. They treat 1 set with sodium bisulfite, which deaminates nonmethylated cytosine to uracil yet does not affect methylated cytosine. 2.) PCR is conducted and the uracil's are turned to cytosine. 3.) The two sets are sequenced and can be compared to determine methylation patterns.

making cDNA Microarrays

1.) Select the the numbers and types of probes for your gene of interest. cDNA clones are generated using plasmid library and sequences are available from the expressed sequence tag (EST). 2.) PCR is used to amplify the cDNA then is denatured. 3.) A robot is used to create the chip for the array. Purified samples of each DNA are put into small wells arranged in a grid in microtiter plate. 3.) UV light is used to cross link the DNA thymine to the glass slide. Note: in newer cDNA microarrays: samples are spotted onto a glass slide using inkjet printer technology

Using microarrays to monitor gene expression +

1.) scientists extract RNA from a cell sample 2.) fluorescently tag the RNA 3.) expose the RNA to the microarray Hybridization of mRNA to DNA probes indicates the degree of gene expression. The level of fluorescence at each point on the array correlates with the level of the corresponding mRNA in the sample.

If you have a genome that is 2 million (2,000,000) bp in size. and you use a lambda vector that is 20kb (20,000) and you want 10x coverage, how many clones do you need? +

20,000 / 2,000,000 = 100 clones x 10 = 1000 clones

Contig

A contig is a contiguous length of genomic sequence in which the order of bases is known to a high confidence level.

Scaffolding

A scaffold is a portion of the genome sequence reconstructed from end-sequenced whole-genome shotgun clones. Scaffolds are composed of contigs and gaps.

Whole genome tiling array (and what it can be used for)

A type of oligonucleotide microarray that has probes that cover the whole genome. They can be used to identify transcription factor binding sites, regions of methylation, SNPs, VNTRs, and repetitive elements. This is useful as finding genetic variations and polymorphisms is critical to genome analysis.

Chromatin Immunoprecipitation (ChIP) <

An example of WGA application. This procedure determines if proteins can binds to or is localized to a particular region of DNA in the chromatin of living cells. 1.) ChIP begins by cross-linking all the various transcription factors to chromatin. 2.) Next, the chromatin is sheared into smaller fragments, and the DNA/transcription factor complexes are isolated. 3.) Finally, the DNA sequences that are bound to the chosen transcription factor are identified using WGA. The entire procedure, including the analysis on a gene chip, is called ChIP-chip

Marfan Syndrome

An example of a mendelian disease. It is associated with a mutation on the fibrillin-1 gene which increases the amount of TGFB leading to abnormal tissue growth. (weakened collagen in the aorta= aortic aneurisms) Doctors can administer TGFB inhibitors that allow for normal tissue production in patients with Marfan Syndrome.

Human Genome Project

An international collaborative effort to map and sequence the DNA of the entire human genome. Considered the greatest achievement of bioinformatics.

luciferase

Another popular reporter gene is luciferase, which emits a pulse of visible light when the correct substrate, luciferin, is supplied (Fig. 8.31). Luciferase is an enzyme encoded by the lux gene in bacteria or the luc gene in fireflies. The two luciferases are not related and have different enzyme mechanisms. Both genes work well as reporter genes and have been cloned onto vectors that work in a variety of different organisms. Detecting the light emitted by luciferase is difficult because of its low levels and requires special equipment such as a luminometer or scintillation counter

taxonomic approach

Classifies organisms based on physical characteristics (phenotype). Doesn't work for organisms such as bacteria. However, molecular phylogenetics allows making family trees for every organism.

Constitutive vs Facultative heterochromatin

Constitutive heterochromatin: remains in a condensed state with no chance of expression. Exists to play a role in chromosome structure. ie. Centromeric region and telomeric region of chromosomes Facultative heterochromatin: has the potential for gene expression at some point in development and can be either condensed or decondensed depending on cell type ie. Barr body

Types of epigenetic modifications

DNA methylation: The addition of methyl groups o bases of DNA that can control gene expression long term Histone modification: changes in the structure of histones that make it more or less likely that a segment of DNA will be transcribed RNA interference: siRNA can be inherited Protein-based epigenetics: regulatory proteins with a changed conformation are inherited and are ultimately responsible for environmental adaptation

Mendelian Diseases

Disease resulting from a single mutation in a single gene and are also heritable. They can be identified using genomics and pedigree analysis.

functional genomics

Functional genomics encompasses the global study of gene expression over the entire genome. Studies: transcriptome: all the RNA transcribed from the genome proteome: all the proteins encoded by the genome metabolome: all the metabolic pathways in the organism

Gene Fusion

Fusing regulatory sequences from an individual gene of interest to a reporter gene allows detailed monitoring of the expression pattern of the gene. Gene fusions are used to monitor genes whose products are difficult to assay. Reporter genes are fused to the regulatory region of the target gene. GENE Fusion: 1-The regulatory region of the gene of interest is isolated first. This segment is normally found upstream of the gene of interest and includes sites for transcription factors to bind, plus various enhancer elements. 2-The coding sequence of the gene of interest is replaced with the reporter gene so that the regulatory elements now control the reporter gene rather than the original gene of interest.

comparative genomics +

If a genome sequence is known, then comparative genomics can be performed. This is a research method that uses similarity searches in a genomic database to discover the functions of unknown genes. This is possible because there are often related sequences with similar functions across many organisms. If a sequence that has an unknown function has an ortholog in another organism, and the function of the gene sequence in the ortholog is known, then the sequences might have the same function. This can apply to more than one gene as often entire pathways are similar between species. (ie insulin signaling proteins in humans and C. elagans.) Comparative genomics cant be relied on fully because proteins with similar sequences may have different roles and functions. Also the databases used are sometimes flawed. Because of this, comparative genomics must be complemented with other studies to accurately discern protein function.

inversions translocations & duplications

Inversions occur when DNA segments become inverted relative to the original sequence. Translocations occur when DNA segments are moved to new locations. Duplications occur when the DNA segment is copied and then moved, resulting in two identical regions.

shotgun sequencing +

It would be possible to sequence a genome by cloning it into many thousands of small plasmids, sequencing these at random, and assembling the reads without knowing the locations of the clones in the genome *The DNA fragments were prepared by breaking genomic DNA mechanically into suitable size, cloned into vectors to make subclone libraries. *It is important to coverage the whole genome, that is the number of independent subclones that will be needed to ensure having a complete sequence. To ensure that most of a genomic is represented in the sequence data, typically a level of 6X - 10X coverage is needed. Genomic DNA is isolated and randomly fragmented by sonication, nebulization, or hydrodynamic shearing. The frayed DNA fragments are repaired and phosphorylated. Fragments are separated into small, medium, and large fractions and cloned into plasmids and fosmids vector. After transformation of E. coli with the library, colonies with cloned DNA are picked and grown. Vectors DNA is purify from each library and sequenced.

LINE vs SINE structure

LINE elements: contain an internal promoter for RNA polymerase II and two open reading frames that encode proteins. The first protein has an unknown function. The second is a bifunctional protein with reverse transcriptase and DNA endonuclease domains. SINE elements: usually contain only an internal promoter for RNA polymerase III and some sort of tRNA stem-loop structure, followed by a poly(A) tail.

Chromosome walking

Method to fill in gaps in genome by adding a probe that is complimentary to the end of known library clone 1. The probe screens the library library, and a second clone is identified. The two clones overlap and are linked to extend the known sequence. this is repeated till the gap is closed.

WGA to determine methylation <

Methylation controls gene expression and cancerous cells have different methylation patterns than normal cell. So scientists can use WGA to look for cancer cells. 1.) Scientists treat genomic DNA with sodium bisulfite, which deaminates nonmethylated cytosine to uracil yet does not affect methylated cytosine. 2.) The treated DNA is then hybridized to a WGA. Those regions with nonmethylated cytosine no longer hybridize to the array because the cytosines have been converted to uracil (which pairs with A, not G). 3.) Those regions of the genome that are methylated still hybridize well because methylated cytosine and guanine form a stable base pair.

Bisulfite Sequencing of the methylome of higher organisms

Methylation controls gene expression and is important in higher organisms. Methylation of the genome occurs on the cytosine of CpG motifs which are of regulatory regions of genes. Methylation of these motifs usually turns off the genes. Examining the methylome by bisulfide sequencing allows scientists to compare sequences subjected to sodium bisulfite with those unsubjected in order to examine methylation pattern.

Moderately Repetitive Genomic Sequences

Most genes are present only once per haploid genome but some gene products are needed in large amounts so are present in multiple copies within the genomes. They are often clustered together ie. ribosomal RNA genes, Histone & tRNA

Making Oligonucleotide Microarrays

Oligonucleotides are traditionally synthesized chemically directly onto the glass slides similarly to how its done with CPG beads. This is done by combining photolithography with solid-phase DNA synthesis.

Base Substitution Mutation

One nucleotide is exchanged for another Transition: purine base is replaced by another purine, or a pyrimidine is replaced by another pyrimidine Transversion: If a pyrimidine is exchanged for a purine, or vice versa Missense: When the base substitution alters one amino acid in a protein Nonsense: A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein

Lac Z gene*

One of the most widely used reporters is the lacZ gene from E. coli, which encodes the enzyme β-galactosidase. This enzyme splits disaccharide sugar molecules into their monomers but also cleaves various artificial substrates. When the substrate ONPG is cleaved, one of the cleavage products forms a visible yellow dye. When X-Gal is cleaved by β-galactosidase, one of the products reacts with oxygen to form a blue dye. FIG 8.29*

pharmocogenetics

Pharmacogenetics is the study of inherited differences in drug metabolism and response. Some SNPs affect how a person metabolizes a certain drug. When scientists determine what SNP correlates with what drug sensitivity, new patients can be screened and possibly avoid adverse drug reactions (ADRs).

Considerations of hybridization on DNA microarrays

Probe attachment: How the probe is attached to the slide can affect how well the probe DNA and target DNA hybridize, especially for oligonucleotide microarrays because of their short length. Spacer length: When the spacer between the glass slide and oligonucleotide is too short, the oligonucleotides are condensed and not accessible to hybridize. If the spacer region is too long, the oligonucleotides and spacers tangle and fold, preventing optimal hybridization. Medium sized spacers work best. Target fragment length: -If the incoming target fragment is too long it can bind with itself forming hairpins. GC content: -more GC base pairs give stronger hybridization

RNA-Seq placenta example <

RNA seq can be used to examine expression patterns in placenta cells versus several other body tissues. Nearly 300 genes showed significantly greater expression in placental tissue, and several transcriptional regulators were identified that were probably involved in this elevated expression.

Advantages of RNA seq over microarrays +

RNA-Seq is more sensitive and gives more quantitative data in these cases. Conversely, RNA-Seq does have the disadvantages of being relatively expensive and requiring sophisticated data analysis.

Reporter Genes

Reporter genes are genes that enable the detection or measurement of gene expression. They can be fused to regulatory sequences or genes of interest to report expression location or levels. Reporter genes are genes whose products can be readily assayed subsequent to transfection, and can be used as markers for screening successfully transfected cells, for studying regulation of gene expression, or serve as controls for standardizing transfection efficiencies. Reporter genes encode enzymes: such as β-galactosidase, alkaline phosphatase, and luciferase that cleave their substrates to form a visible dye or light. Green fluorescent protein has luminescent properties that allow it to absorb one wavelength of light and emit a longer wavelength.

How Genome gaps arise

Result from heterochromatin, highly condensed repetitive DNA found in specific sites throughout the genome. The physical nature of heterochromatin makes it difficult to sequence. Occurs due to methylation

Markers in Physical Mapping

SNPs, VNTRs, RFLPs, and microsatellites are also used in physical mapping but do not provide enough markers. STS is necessary - STS (sequence tagged sites): short sequences of 100 to 500 base pairs that are unique and can be detected by PCR. - EST (expressed sequence tag): A specialized type of STS that uses a cDNA library. This means that the EST is expressed as mRNA.

RFLP analysis vs STS Mapping Using Restriction Enzyme Digests

STS Mapping can determine the distance between two markers as the number of base pairs, whereas RFLP analysis determines how often two different markers are found together but the actual distance is based on how often its inherited in offspring.

Restriction enzyme mapping (STS physical mapping)

STS mapping is shown for four STS sites on a single chromosome. Restriction enzymes cut it up. The number of times two STS sequences are found on the same fragment reveals the proximity of the two markers. ie the two purple STSs are found on the same fragment six times and must be close to each other. The two green STSs are found on the same fragment only two times and are therefore farther apart. The purple and green STSs are never found on the same fragment so they must be far apart

Whole exome sequencing

Scientists can sequence the exons only instead of the whole genome, this creates a smaller dataset and lowers cost. This sequencing allows the identification of mutations in the sequences that specifically affect proteins, and can suggest whether or not that variation is the causative agent of the disease.

contig mapping

Small clones have regions that overlap with each other. Ordering the small clones into one sequence forms a contig map.

Shotgun Sequencing Overview +

Start with Genomic DNA Cut it up End repair and phosphorylate Separate by Size Ligate, clone, and transform E. coli Culture E coli Extract DNA and sequence Assemble contigs Scaffolding and gap closure Finished sequence

GFP as reporter protein

The gene for GFP has been integrated into the genome of animals, plants, and fungi. After exposure to long-wavelength UV, the organisms emit green light. ex-(A) Transgenic mice with GFP among normal mice from the same litter. The gfp gene was injected into fertilized egg cells to create these mice. The main advantage of using GFP as a reporter is the ability to see expression in living tissues

Figure 8.27 Microarray versus RNA-Seq +

The microarray approach relies on using probes to bind labeled RNA. The level of expression is deduced from the fluorescent signal due to the hybridization of RNA with probe. In contrast, RNA-Seq is independent of probes and relies on directly counting the number of copies of sequenced cDNA derived from the RNA.

How many genes in a genome?

The number of genes in an organism depends on the definition of gene and the distinction between real genes and pseudogenes. The absolute number of genes in any sequence is approximate.

PhoA gene

The phoA gene is another reporter gene that encodes alkaline phosphatase. Alkaline phosphatase removes phosphate groups from various substrates. When the phosphate group is removed from o-nitrophenyl phosphate, a yellow dye is released. When the phosphate is removed from X-Phos, further reaction with oxygen produces an insoluble blue dye. When the phosphate is removed from 4-methylumbelliferyl phosphate, this releases a fluorescent molecule.

Bioinformatics

The scientific discipline of using computers to handle biological information which includes the storage, retrieval, and analysis of data about biomolecules. Examples include analyses of data from DNA microarrays (see later discussion), assessment of the function of genomes, and the comparison of different genomes. It encompasses a large number of subfields listed in the table.

Epigenetics/Epigenome

The study of influences on gene expression that occur without a DNA change. This can be caused by specific environmental or non genetic factors, such as diet and exposure to certain chemicals. The epigenome is the total number epigenetic changes in a genome. Changes are not considered epigenetic unless the altered gene expression state is inherited by another generation of cells (epigenetic inheritance).

Data Mining

The use of computer programs to search and interpret the data. Bioinformatics programs are developed that search the genomic data banks and sift, sort, and filter the raw sequence data. They follow these steps:

RNA-seq +

This technique uses next-generation sequencing to more accurately quantify gene expression. What's occurring is that the RNA is being quantified indirectly by the amount of cDNA sequenced whereas in a standard microarray expression is analyzed by relative fluorescence level. RNA seq is probe independent while standard microarray is probe dependent. It can be used to examine gene expression the similar cells under different conditions or in specialized cells of a higher organisms.

Tiling Arrays

Tiling arrays differ from traditional microarrays in the nature of the probes. Instead of probing for sequences of known or predicted genes that may be dispersed throughout the genome, tiling arrays have the potential to identify new regions whose functions are not known, whether these encode unknown protein-encoding genes or nontranslated RNA.

Two-color cDNA microarray experiment design +

Used to determine the set of genes that are expressed in higher level in different tissue. 1.) Two different sets of complex mRNA sample are used 2.) Each set, a control and the test sample, are labeled separately with a different fluorescent label, usually Cy3 (green) or Cy5 (red.) 3.) The two labeled samples are mixed together and hybridized to the microarray The ratio of the amounts of each mRNA in the experimental to control samples is determined by relative fluorescent level at each spot.

Gene Expression during Yeast Cell Cycle <

Using microarrays, you can see what genes are active at what point in the yeast cell cycle. To do this you culture yeast cells then use α factor or mutant yeast to freeze the cell at particular stages of mitosis. The gene expression patterns for each stage are examined. Color coding can be used to make a chromosome map that shows where genes are active during which segment of the cell cycle.

Somatic vs Germline mutations

When a mutation occurs in the somatic cells, the children or offspring do not inherit the mutation; only when a mutation occurs in the germline, or sex cells, are the mutations passed on to the next generation

SNP (single nucleotide polymorphism)

When found within the coding regions of genes, SNPs may alter the amino acid sequence of the protein. This in turn may affect protein function. i.e., the SNP corresponds to a difference in an actual gene. If a SNP correlates with a genetic disease, identifying that SNP may diagnose the disease before symptoms appear

Gene sequence alignment*

When gene sequences are compared, they are aligned, so that the regions of highest similarity correspond. This type of alignment can determine the relatedness of two or more proteins or genes. Based on alignments, genes have been grouped into families, groups of closely related genes that arose by successive duplication and divergence. Gene superfamilies occur when the functions of the various genes have steadily diverged until some are hard to recognize*

Whole genome tiling array designs

Whole-genome arrays (WGAs): contain oligonucleotide probes that cover the entire genome in an overlapping set. Quasiwhole-genome arrays: the probes are spaced equal distances apart through the genome. The probes thus cover the entire genome, except for the gaps between the probes. Splice junction arrays: have only probes that span the upstream and downstream regions of known splice junctions in mRNA. Exon-scanning arrays: have probes derived from exon sequences only

VNTR (variable number of tandem repeats)

aka minisatalites. tandem (next to each other) repeats of a short DNA sequence (9 to 80 base pairs). The number of repeats differs from one person to the next; therefore, these can be used as specific markers on a genetic map.

microsatellite polymorphism

also a tandem repeat. However, unlike VNTRs, microsatellites are repeats of 2 to 5 base pairs in length.

cladistic approach

assumes that any two organisms ultimately derive from the same common ancestor and that at some point bifurcation, or separation into two clades, occurred in their line of descent

2 Types of DNA microarrays

cDNA: fragments 600 to 2400 nucleotides in length oligonucleotide: fragments of 20 to 50 nucleotides in length

Advantage of cDNA probes for hybridization

cDNAs are double-stranded, so secondary structures such as hairpins are less likely to be a problem. Note that cDNA arrays must be denatured either with heat or chemicals before hybridization.

RFLP (restriction fragment length polymorphism)

differences in DNA fragment sizes cut by specific restriction enzymes, which are used as markers on genetic maps, to help identify the source of DNA and to determine the presence of specific alleles or genes

Polygenic Diseases

diseases that are caused by a variety of genes, not one specific mutation.

radiation hybrid mapping, cytogenetic mapping, and FISH?

dont have to know the specifics

genetic marker

genetic indicators that are perceivable things that can be tested. 3 Types: - genes - biochemical traits - DNA markers such as RFLPs, VNTRs, microsatellites and, SNPs. For example, a gene that has an allele that produces a detectable phenotypic difference can be observed in an individual. Or an RFLP can be detected using restriction analysis with gel electrophoresis.

Essay Question: Explain two color cDNA microarray, how to interpret the results and the application +

https://www.youtube.com/watch?v=6ZzFihESjp0&t=28s&ab_channel=FaisalFrezeePrime slide 42 - 45 / not in textbook

Using microarrays to monitor cells grown under a variety of conditions

ie heat shock, acid exposure, cancer, or other disease states - lets say that there are 2 conditions. Each condition results in the expression of a gene to adapt to the condition. - Under condition 1, a certain RNA is expressed which is labeled with red fluorescence. - under condition 2, RNA is expressed which is labeled with green fluorescence. - By looking at a microarray, you can see what genes were expressed. if a cell was under both conditions then you would see yellow.

Essay Question: Compare and contrast microarray and RNAseq +

page 280 / slide 48

Phase Variation of the PapAB Pilus

papBA operon of uropathogenic Escherichia coli encodes the pyelonephritis-associated pilus that allows it to bind to host kidneys cells causing UTI's. Synthesis of the pilus is determined by the operon and it flips back and forth from of to off depending on methylation. - The promoter region of the operon has two clusters of binding sites for the Lrp protein. Each of these clusters may be methylated or nonmethylated. - The Lrp protein will only bind to nonmethylated DNA. When Lrp binds close to the promoter (sites 4, 5, and 6), transcription is blocked. - PapI protein makes the switch and changes methylation so Lrp can bind to sites 1, 2, and 3 and the operon is turned on.

Genome Map

provide a linear series of landmarks for use when putting together sequence data. Two Types - Genetic mapping: by observing the offspring result of mating crosses or pedigree maps, you can observe 2 different markers. If genes are linked close together, they will be inherited together more often. You can then calculate the genetic map unit distance between genes by looking at the percentage of offspring that inherit both markers. Genetic mapping does not give the exact bp distances though, just relative location. - Physical maps: give the distance between markers in base pairs because the markers are physically associated with a location on a chromosome.

DNA Mutations

table

insertion or deletion mutation

the addition or loss of one or more bases to the DNA. Can cause frameshifts

phylogenetic trees

used to graphically represent the relatedness of organisms. features: a root, nodes, and branches. Root represents the common ancestor. Individual nodes represent common ancestors between two subgroups of organisms. Branches represent clades( groups of organisms with a common ancestor). The length of the branches indicates the number of sequence changes, so if the branches are short, the two groups of organisms bifurcated relatively recently, and if the branches are long, the split occurred long ago.

Molecular phylogenetics

uses genomic sequences of different organisms to determine their evolutionary relatedness. Essential proteins have fewer mutations over time. Less essential proteins have more mutations over time.

Zipcode analysis

zipcode region: region that is used to identify a specific SNP, and each SNP has a different zipcode 1.) PCR aplifies regions containing each SNP being investigated 2.) single base extension analysis where a zipcode primer is designed to anneal 1 bp away from the SNP location. 3.) DNA polymerase plus fluorescently labeled ddNPT are added resulting in single bp added to the primer. Each base is labeled with a different fluorescent dye, allowing it to be identified. 4.) beads linked to complementary zipcode sequences are added to grab the zipcoded primers. 5.) FAC is used to sorted and count the beads, determining the identity of the nucleotide in the patient.