Ch 21

Cytogenetic mapping

- Also called cytological mapping - Relies on light microscopy - Genes are mapped relative to band locations

The DNA sequences used in microarrays can be from what sources?

- Clones - PCR products - synthetic sequences

shotgun sequencing

- Clones from genomic or chromosomal library isolated randomly and sequenced, possibly multiple times. - These overlapping sequences are matched together using computers. - This is how the human genome was mapped.

Molecular markers

- DNA sequences that do not encode genes. - Markers need to be polymorphic(show variation between at least some individuals in a population)

What are some of the uses of metagenomics?

- Development of human medicine - Improving Agricultural yields by examining soil microorganisms. - Bioremediation- Microorganisms in water and soil can be used to decompose pollutants in environment.

Sequencing by synthesis(SBS)

- Each nucleotide is identified immediately after being incorporated to the growing DNA strand. - If a nucleotide is incorporated into a DNA strand, pyrophosphate is released and detected by the computer.

DNA microarrays can be used for?

- Finding genetic variations - Cell-specific gene expression - Gene regulation - Tumor profiling - Microbial strain identification

What are advantages of RNA seq over microarrays?

- More accurate at quantifying amount of each transcript. - Better at detecting low abundance RNAs. - Identifies boundaries between exons and introns. - Identifies 5' and 3' ends of RNA transcripts and aids in identifying transcriptional start sites.

What are contigs?

- Pieces of chromosomal DNA that have been broken with restriction enzymes. - Clones are then placed into vectors to create genomic DNA library. -Organize the pieces according to their location on the chromosome.

Physical Mapping

- Relies on DNA cloning techniques - Genes are mapped relative to each other - Distances computed in number of base pairs

Linkage Mapping

- Relies on genetic crosses - Genes are mapped relative to each other - Distances computed in map units( or centiMorgans)

What can a DNA microarray be used for

- finding genetic variations - cell-specific gene expression - Gene regulation - Tumor profiling - Microbial strain identification

How does Chromatin Immunoprecipitation (ChIP) work?

- utilizes proteins cross linked to DNA - DNA then isolated from cells and broken into smaller pieces. - Antibody is used to precipitate the protein-DNA complexes. - DNA is then purified and amplified with PCR. - The sequence of the amplified DNA can be identified by using it as a probe on a microarray.

What two things are most responsible for the proteome being larger than the genome?

1) Alternative splicing - mRNA can be spliced to code for different combinations of amino acids. 2) Posttranslational covalent modification - Irreversible changes: Proteolytic processing, disulfide bonds, attachment of prosthetic groups - Reversible changes:

What are the steps of shotgun sequencing?

1) Break open cells to expose DNA and then remove aqueous layer containing DNA. 2) Sonicate the DNA to break it into fragments. 3) Clone DNA fragments into vectors to produce DNA library. 4) Subject clones to dideoxy DNA sequencing. 5) Use bioinformatics to identify various types of genes in the genome.

How is fluorescence in situ hybridization(FISH) carried out?

1) Cell are treated with agents to swell them and to fix them to a slide. 2) Chromosomal DNA is denatured. 3) A DNA probe is added. This probe should be complimentary to a specific gene of interest. 4) The location of the probe on the chromosome can then be determined because it is labeled with a fluorescent molecule which lights up when looked at under a fluorescent microscope. 5) The results of FISH are then compared with chromosomes marked with geimsa stain and the location of the probe can now be mapped relative to the banding.

RNA sequencing allows transciptomes to be compared in?

1) Different cell types 2) Healthy vs. Diseased cells 3) Different stages during development 4) Different environmental conditions

What are the two types of postranslational covalent modifications?

1) Irreversible: these changes may be necessary to produce a functional protein. (Proteolytic processing, disulfide bonds, attachment of prosthetic groups(sugar or lipids)). 2) Reversible: changes that transiently affect protein function.(Phosphorylation, methylation, acetylation)

The two categories used to identify features in bioinformatics fall into what two groups?

1) Sequence recognition - program identifies specific sequences 2) Pattern Recognition - Does not rely on specialized sequence information - Program identifies a pattern of symbols that can occur within any group of symbol arrangements.

How can eukaryotic chromosomes be distinguished?

1) Size 2) Banding 3) Location of centromeres

What methods can be used to align members of a contig?

1) Southern blotting 2) Use of molecular markers( such as STSs) 3) Analysis of restriction enzyme digests.

What does the name contig mean?

A Contiguous region of a chromosome that is found as overlapping regions within a group of vectors.

Chromosome Walking

A DNA mapping technique that begins with a gene or other sequence that has already been cloned, mapped, and sequenced and "walks" along the chromosomal DNA from that locus, producing a map of overlapping restriction fragments.

What are Gene Knockout collections?

A collection of Animals or organisms that has a gene specifically inactivated. - These can be made using transposable elements or CRISPR-Cas technology.

What are microsatellites?

A site in the genome that contains many short sequences that are repeated many times in a row. Microsatellites can also be polymorphic from individual to individual within a species. Microsatellites are isolated using PCR. They are also called short tandem repeats(STRs) or simple sequence repeats(SSRs)

To map the distance between molecular markers via testcrosses, the markers must be? A) Polymorphic B) Monomorphic C) fluorescently labeled D) on different chromosomes

A) Polymorphic

Shotgun sequencing is a method of DNA sequencing in which A) the DNA fragment to be sequenced are randomly generated from larger DNA fragments. B) the sequencing reactions are carried out in rapid succession. C) the samples to be sequenced are rapidly generated by PCR. D) all of the above

A) the DNA fragment to be sequenced are randomly generated from larger DNA fragments.

A gene knockout is a gene A) whose function has be inactivated. B) that has been transferred to a different species. C) that has been moved to a different location in the genome. D) that has been eliminated from a species during evolution.

A) whose function has be inactivated.

To study gene expression in a particular sample of cells, what material is usually hybridized to a DNA microarray? A) Fluorescently labeled mRNAs B) Fluorescently labeled cDNAs C) Radiolabeled mRNAs D) Radiolabeled cDNAs

B) Fluorescently labeled cDNAs

What are bioinformatics and their use?

Bioinformatics is the use of computers to analyze data It can be used for finding/understanding: - gene structure - gene function - relationship between genes and organisms - protein interactions

Metagenomics is aimed at? A) determining the complete genome sequence of a newly identified microorganism. B) mapping the genes on chromosomes of newly identified microorganisms. C) determining the sequence of DNA fragments in environmental samples. D) determining the functions of all the genes in a given species genome.

C) determining the sequence of DNA fragments in environmental samples.

A DNA microarray is a slide that is dotted with A) mRNAs from a sample of cells. B) fluorescently labeled cDNAs. C) known sequences of DNA. D) known cellular proteins.

C) known sequences of DNA.

The purpose of a chip-chip assay is to determine? A) the expression levels of particular genes in a genome. B) the amount of a specific protein that is made in a given cell type. C) the sites in a genome where a particular protein binds. D) all of the above.

C) the sites in a genome where a particular protein binds.

What type of chromosome mapping relies on light microscopy?

Cytogenetic mapping. This uses stains and a microscope to identify chromosomes and maps their genes based on relative banding location.

Which of the following is an example of a molecular marker? A) RFLP B) Microsatellite C) Single-nucleotide polymorphisms D) All of the above

D) All of the above

Why is the proteome larger than the genome?

Due to alternative splicing, RNA editing, and post-translational modification

What are identical genes from different species called?

Homologs. These are genes that are derived from the same ancestral gene.

In shotgun sequencing, after the cells are broken open and exposed by adding phenol and chloroform, where is the DNA located?

In the aqueous layer.

How accurate is separating a mixture using two dimensional gel elecrophoresis?

It can resolve two proteins that differ by a single charged amino acid.

The proteome is ______________ than the genome.

Larger

What type of chromosome mapping relies on crosses?

Linkage mapping. By looking at the offspring of two heterozygotes you can use the number of recombinant offspring to measure the distance between genes.

What are the three main phases of genomic analysis?

Mapping Sequencing Functional Genomics -Analysis of gene interactions - Includes proteomics- how proteins interact

How is an amino acid sequence determined after running a two dimensional gel?

Mass Spectrometry

DNA microarrays (DNA chips)

Monitors the mRNA expression of thousands of genes simultaneously.

What is the ultimate goal of physical mapping?

Obtain a complete contig for each type of chromosome.

In pyrosequencing what are attached to the ends of DNA fragments?

Oligonucleotide adapters are attached to both the 5' and 3' ends.

When two homologous genes are found in a single organism, these genes are termed?

Paralogs

Microsatellites can also be used in _____________________ ___________________.

Pedigree analysis.

What type of chromosome mapping relies on DNA cloning or DNA sequencing techniques?

Physical mapping This involves cloning and vectors to examine the distance between genes. Distances are given in the number of base pairs between genes.

What are microsatellites?

Repeating sequences of noncoding DNA whose integrity is maintained in cell division. - Number of microsatellite repeats at a locus is variable.

A molecular marker is a __________________ that is found at a specific site on a chromosome and has properties that allow it to be _____________.

Segment of DNA uniquely identified using molecular tools.

Positional cloning

Strategy to clone a gene based on its mapped position along a chromosome. - This has been successful in cloning many human genes, especially those that are disease causing.

Would you expect the amino acid sequence or the nucleotide sequence to be more highly conserved?

The nucleotide sequence. The nucleotide sequence is what codes for the amino acids. I believe this sequence would be more conserved because the amino acid sequence has already found a way to adapt to mutations/errors with the "Wobble base". You can also see how conserved the nucleotide sequence is by looking at the genes of different species where oftentimes you can find identical genes preserved through millions of generations and through different species.

What technique can be used to separate a complex mixture of proteins?

Two intentional gel electrophoresis

In order to create contigs of eukaryotic chromosomes researchers have to?

Use artificial vectors which can hold large segments of chromosome. - Yeast Artificial chromosomes - Bacterial artificial chromosomes - P1 artificial chromosomes(PACs) - Phage hybrids are cosmids

Sequence tagged site(STS)

When a pair of primers amplifies a single site within a set of chromosomes this site is known as an STS.

Can you use multiple probes when performing In situ hybridization?

Yes. Probes only bind to specific sequence and the probes can all be labeled with a different color. -Also called Chromosome painting

Chromatin Immunoprecipitation is used in conjunction with a microarray this is called?

a ChIp-chip assay

RNA sequencing (RNA-seq)

a method of analyzing large sets of RNAs that involves making cDNAs and sequencing them

What is a molecular marker?

a segment of DNA that is found at a specific site along a chromsome and has properties that enable it to be uniquely recognized using molecular tools.

PCR amplification is used to?

amplify specific microsatellites.

Mass spec can also be used when trying to determine if a protein has?

any covalent modifications such as phosphorylation. It can detect the mass of a single phosphate which will show up in the final mass of the spec.

Chromatin Immunoprecipitation (ChIP)

can determine if proteins can bind to a particular region of DNA in the chromatin of living cells. - Proteins are crossed-linked to DNA - DNA isolated from cells and broken down into small pieces. - Antibody is used to precipitate the protein-DNA complexes. - DNA is purified and amplified with PCR. - Sequence of the amplified DNA can be identified by using it as a probe on a microarray.(ChIp-chip assay)

In new DNA sequencing technologies there is no need to?

clone DNA. - Can be sequenced from a mixture of fragments.

Physical mapping involves?

cloning of DNA from a chromosome. - Characterize the clone for size, genes it contains, and their relative positions. -Match a piece of cloned DNA to another piece of cloned DNA and repeat process until all DNA of interest can be found on clones that overlap. - This creates a contig

What is the goal of Functional Genomics?

elucidate the roles of genetic sequences in a given species.

The technique of fluorescent in situ hybridization involves the use of a ______________ that hybridizes to a __________________.

fluorescent probe, specific site on an intact chromosome.

What are molecular markers good for?

good for DNA fingerprinting

Chromatin Immunoprecipitation (ChIP) is used to determine?

if proteins can bind to a particular region of DNA.

DNA microarrays assess what?

mRNA and gene expression. It is called DNA microarray because it is DNA that is spotted on the plate.

DNA microarrays are used to asses what?

mRNA. It is called DNA microarray because its DNA that gets spotted onto the plate.

When two homologous genes are found in different species, these genes are termed?

orthologs

Linkage mapping relies on the frequency of?

recombinant offspring to map genes

Microarrays use what to change mRNA to be assessed?

reverse transcriptase

metagenomics (environmental sequencing)

the inventory of all the genes in a community or ecosystem by sequencing, analyzing, and comparing the genomes of the component organisms from that environment.

What is the isoelectric point?

the pH at which the molecule is electrically neutral

Pyrosequencing relies on?

the release of pyrophosphate.

Proteomics

the study and comparison of all the proteins that result from an organism's genome and how they interact.

Metagenome

the total genetic complement of all the cells present in a particular environment.

In situ hybridization

uses fluorescent dyes attached to probes to identify location of specific DNA sequences in place in intact organism