Chapter 22

uses of metagenomics (3)

1. Human medicine Metagenomics is used to characterize populations of microorganisms in the mouth and intestines, and find changes associated with disease 2. Agriculture Metagenomic analysis of soil samples reveals astonishing complexity of soil microorganisms, which may improve agricultural yields 3. Bioremediation Microorganisms in soil and water help decomposition of pollutants in the environment Metagenomics may identify useful microorganisms

Physical mapping involves 1. _____________ a piece of DNA from a chromosome 2. Characterizing the clone for its __________ 3. Matching a piece of the cloned DNA to another cloned DNA and repeating the process until all DNA of interest can be found on clones that overlap -------This creates a _____ Ultimate goal of physical mapping is to obtain a complete ____ for each type of chromosome

1. cloning 2. size, genes that it contains, and their relative locations contig contig

Types of Chromosome mapping

Cytogenetic mapping Linkage mapping Physical mapping

Physical mapping Relies on _____ Genes are mapped relative to ______ Distances computed in ______

DNA cloning techniques each other number of base pairs

________- locate the position of a gene or other unique DNA segment in a chromosome Researchers use a probe to detect the "target" DNA: __________ uses fluorescently labeled DNA probes

In situ hybridization Fluorescence in situ hybridization (FISH)

_______ relies on the frequency of recombinant offspring to map genes DNA sequences that do not encode genes can also be mapped relative to one another, such sequences are known as _______ ----There are several types of DNA sequences commonly used as molecular markers Molecular markers need to be ______ - show variation between at least some individuals in a population ------Similar to different alleles of a gene ------Good for DNA fingerprinting

Linkage mapping molecular markers polymorphic

______ markers are identified using PCR _____ Short, simple sequences Abundantly dispersed throughout a species' genome Number of microsatellite repeats at a locus ___________ Example: _____ ---The most common human microsatellite ---n may range from 5 to 50+ ---Found about every 10,000 bases

Microsatellite Microsatellites is variable (CA)n

_________ is used to amplify specific microsatellites When a pair of primers amplifies a single site within a set of chromosomes the region amplified is called a ______________ Since the number of times the microsatellite sequence is repeated may vary, can get PCR products of different _____ even from the same individual

PCR amplification sequence tagged site (STS) lengths

_____ - strategy to ___________ a gene based on its mapped position along a chromosome -----Been successful in the cloning of many human genes, especially those that are disease-causing Examples: A common method used in positional cloning is ______ --A gene's position relative to a marker must be known ----This provides a starting point to molecularly "walk" toward the gene of interest

Positional cloning clone examples: Cystic fibrosis, Huntington disease, Duchenne muscular dystrophy chromosome walking

__________________ ---Does not require an extensive physical map ---Faster and easier ---Clones from a genomic or chromosomal library isolated randomly and sequenced, possibly multiple times ---Overlapping sequences matched together using computers ___________________________________

Shotgun sequencing used to map human genome

--Different experimental strategies can be used to align the members of a contig: 1. 2. 3. --Geneticists can also correlate cloned DNA in a contig with markers obtained from linkage or cytological methods

Southern blotting Use of molecular markers (such as STSs) Analysis of restriction enzyme digests

To create contigs of eukaryotic genomes, the cloning vectors have to accept large chromosomal fragments 1. ________ Inserts can be several hundred thousand to 2 million bp long 2._____ Inserts can be up to 300,000 bp long 3._______-phage hybrids are cosmids Inserts can be up to 300,000 bp long

Yeast artificial chromosomes (YACs) Bacterial artificial chromosomes (BACs) P1 artificial chromosomes (PACs)

The fluorescent probe is seen as a colorfully glowing region or spot Probe will only bind a specific sequence --Multiple probes can be used at one time ----Each probe labeled with a different dye that emits a different color ----Called _______ Results of a FISH experiment can be compared to ______ chromosomes Location of a probe can be mapped relative to the ______

chromosome painting Giemsa-stained G banding pattern

The _____________ consists of a series of subcloning and library screening experiments In ________ a small piece from the end of one clone is inserted into another vector It is then used as a probe in a library screen to isolate the next clone Process is repeated until the DNA of interest is reached

chromosome walk subcloning

Physical mapping --Involves the ______ of many pieces of chromosomal DNA --Used to actually isolate DNA that contains gene of interest --Very labor and time intensive - takes a collaborative effort --In recent years, physical mapping studies have led to the DNA sequencing of entire genomes

cloning

CONTIGS A sample of chromosomal DNA is digested into many smaller pieces with restriction enzymes -----Fragments are cloned into vectors to create a genomic DNA library The next step is to organize the chromosome pieces according to their exact location on a chromosome ----DNA is digested so that the clones contain overlapping pieces of chromosomal DNA This collection of clones is known as a _____________ -----It contains a _____ region of a chromosome that is found as overlapping regions within a group of vectors

contiguous contig

Cytogenetic mapping Also called _____ Relies on _____ Genes are mapped relative to _____

cytological mapping microscopy band locations

Genetic map or chromosome map A chart showing relative locations of ____ or other _________ _______ - site where specific genes or other DNA segments are located

genes DNA segments on chromosome Locus (pl. loci)

Linkage mapping Relies on _____ Genes are mapped relative to ____ Distances computed in _____

genetic crosses each other map units (or centiMorgans)

The term ______ refers to the total genetic composition of an organism ______ is the molecular analysis of the entire genome of a species Three main phases of genomic analysis: 1. 2. 3. ----Analysis of gene interactions Includes _____ - how proteins interact

genome Genomics Mapping Sequencing Functional genomics ----proteomics

Cytogenic mapping used to determine ____ of a particular gene relative to a banding pattern Cytogenic mapping relies on light __________________ --Fairly crude limit of resolution --Most species ~ 5 million bp Resolution is much better in species that have ____ chromosomes

location polytene microscopy

--Cytogenic mapping relies on _____________________ Commonly used with eukaryotes which have much larger chromosomes --Eukaryotic chromosomes can be distinguished by: 1. 2. 3. --Chromosomes are treated with particular dyes The banding pattern that results is used for mapping

microscopy Size Location of centromeres Banding patterns

Innovations in DNA Sequencing New technologies have helped to: ---Increase speed of sequencing ----Make large sequencing projects less expensive ______________________DNA can sequence from a mixture of fragments --Automation ---________ - many sequences run in parallel ---Improvements in how sequencing performed -______, sequencing by synthesis

no need to clone High-throughput sequencing pyrosequencing

Microsatellites can be used in _______ Follow inheritance of microsatellite with another trait Identify regions that might have a gene that causes trait

pedigree analysis

Genome sequencing projects --The clones from a physical map can be used for sequencing --All the clones that are sequenced can be used to generate the sequence of a chromosome as their relationship is known ---Whole genomes have been sequenced using these techniques ---But an easier and faster method is _______

shotgun sequencing

Human Genome Project 1995 - researchers led by Craig Venter and Hamilton Smith published the first complete genome sequence of an organism, ______________ 1996 - Consortium of more than 100 laboratories publishes first genome sequence of a ____________ Their successes fueled the pursuit of the human genome project

the bacterium Haemophilus influenzae eukaryote, Saccharomyces cerevisiae

Most microbes in the world are "_______" - they cannot be grown in the lab ______ showed that environmental samples had a huge variety of microbial genes that could be PCR'd from unculturable microbes ____________________ - the study of a complex mixture of genetic material obtained from an environmental sample _______ - a collection of genes from a particular environmental sample -----Can be analyzed similarly to the study of a single genome

unculturable Norman Pace metagenomics Metagenome