Bio 325
The most common micro satellite or STR is ___ where n is anywhere between 5-50 repeats.
(CA)n
BACs and PACs were developed from bacterial F factors and P1 bacteriophage chromosomes, respectively.
.
What are the three ways to determine the organization of DNA regions?
1) cytogenetic mapping 2) linkage mapping 3) physical mapping
The micro satellite is found about every ______ bases in the human genome.
10,000
YACs can accept chromosomal DNA of several hundred thousand to __ ______ base pairs.
2 million
What is structural genomics?
3-D structures of every protein encoded by a given genome.
What is a pico titer plate?
A flat plate with multiple wells used as small test tubes.
What is physical mapping?
A third approach is physical mapping in which DNA-cloning techniques are used to determine the location of and distance between genes and other DNA regions. In a physical map, the distances are computed as the number of nucleotide base pairs between genes.
Describe the procedure for RFLP.
Analyze the DNA by gel electrophoresis after it has been exposed to a restriction enzyme that cuts the arrows. Restriction fragments that vary in length at the chromosomal site from 2 different individuals. Analyze the DNA by gel electrophoresis after it has been exposed to a restriction enzyme that cuts at the arrows.
What is linkage mapping?
By comparison, we considered how genetic crosses are conducted to map the relative locations of genes within a chromosome. Such genetic studies, which are called linkage mapping or genetic mapping, use the frequency of genetic recombination between different genes to determine their relative spacing and order along a chromosome.
What is a cosmic?
Cosmids accept smaller pieces of DNA. A cosmic is a hybrid between a plasmid vector and a phage lambda.
Describe the process of physical mapping.
Cut the YAC vector with EcoRI and BamHI. Cut the chromosomal DNA with a low concentration of EcoRI to yield very large fragments. Mix and add DNA ligase. Large piece of chromosomal DNA integrated into the chromosome.
What is cytogenetic mapping?
Cytogenetic mapping (also called cytological mapping) relies on the localization of gene sequences within chromosomes that are viewed microscopically. When stained, each chromosome of a given species has a characteristic banding pattern, and genes are mapped cytogenetically relative to a band location.
_____ individuals have two copies of a given STS. When an STS contains a micro satellite, the two PCR products will be identical if the region is the same length in both copies (i.e. if the individual is homozygous for the micro satellite). If an individual has two copies that differ in the number of repeats in the micro satellite sequence (i.e. if the individual is heterozygous for the micro satellite), the two PCR products will be slightly different in length.
Diploid
What is genomics?
Genomics is the study of all the genetic material of an organism.
__ _____ _______ is widely used to cytogenetically map the locations of genes and other DNA segments within large eukaryotic chromosomes.
In situ hybridization
Describe the process of pyrosequencing, a next-generation sequencing technology.
Isolate genomic DNA and break into 300- to 800- bp fragments. Covalently attach oligonucleotide adapters to the 5' and 3' ends of the DNA. Denature the DNA into single stands. The mixture of single stranded DNA fragments with adapters is called the sample library. Attach the single stranded DNA fragments to beads via the adapters. Initially, just one DNA strand is attached to a bead. Emulsify the beads to an oil-water mixture so that there is only one bead per droplet. The droplets also contain PCR reagents that amplify the DNA until each bead contains about one million copies of a particular DNA segment. Deposit the beads into a pico titer plate. Only one bead can fit into each well. Add sequencing reagents, which include primers complementary to the adapters. The synthesis of DNA is monitored in real time. The method is referred to as sequencing by synthesis (SBS), , because it involves the identification of each nucleotide immediately after its incorporation into a DNA strand by DNA polymerase. Pyrophophate is released when the nucleotide is incorporated into the growing strand. Pyrophosphate + adenosine 5' phosphosulfate in the presence of ATP sulfurylase produces ATP. ATP + luciferin in the presence of luciferase produces light, and the light is detected by a camera in the sequencing machine. Therefore, light is given off only when a nucleotide is incorporated into a DNA strand. By sequentially adding solutions with only one of the four possible nucleotides, the sequence of the DNA strand can be monitored in real time by deterring when light is given off.
________ or short tandem repeats are short repetitive sequences abundantly interspersed throughout a species genome; they are quite variable in length among different individuals.
Microsatellites
What is functional genomics?
One a genome sequence is known, researchers can examine, at the level of many genes,how the components of genome interact to produce the traits of an organism. This approach is called functional genomics.
Second generation sequencing platforms use ___ to amplify the DNA.
PCR
Using ___ ______, a particular micro satellite among the thousands in the genome may be amplified. This is called a sequence-tagged site (STS).
PCR primers
Describe the process of PCR primer use.
PCR primers complementary to a particular micro satellite's unique flanking segments are added to a set of chromosomes where they specifically recognize and bind to the flanking sequences. Many cycles of PCR produce a large amount of the DNA fragment contained between the two primers.
What is physical mapping?
Physical mapping is an approach n which DNA-cloning techniques are used to determine the location of an distance between genes and other DNA regions. In a physical map, the distances are computed as the number of nucleotide base pairs between genes.
______ ______ ______ ____ results when chromosomal DNA from different individuals is digested with a restriction enzyme and the individuals differ in the number of palindromes they have for that particular restriction enzyme.
Restriction Fragment Length Polymorphisms (RFLPs)
What is an organism's genome?
Set of DNA of an organism.
What is the oak of cytogenetic mapping?
The goal of cytoenetic mapping is to determine the location of genes along an intact chromosome.
What is proteomics?
The goal of researchers is to determine the roles of all cellular proteins, as well as the interactions that these proteins experience, to produce the characteristics of particular cell types and the traits of complete organisms.
Describe the process of cytogenetic mapping.
Treat cells with agents that make them swell and fixes them onto slide. Denature the chromosomal DNA. Add single-stranded DNA probes that have biotin incorporated into them. Add fluorescently labeled avidin that binds to biotin.
What is linkage mapping?
We consider how genetic crosses are conducted to map the relative locations of genes within a chromosome. Linkage mapping or genetic mapping use the frequency of genetic recombination between different genes to determine their relative spacing and order along a chromosome.
What (3) vectors are used to clone large segments of DNA?
YAC, BAC, AND PAC vectors
What are YACs?
Yeast artificial chromosomes.
RFLPs may be mapped and used as _______ ________.
chromosomal markers
Newer methods of DNA sequencing no longer require the _____ of DNA into vectors.
cloning
Physical mapping requires the _______ of many pieces of chromosomal DNA.
cloning
In chromosome walking for positional cloning under the category of physical mapping, make a probe that is _______ to the end of the known gene and use the complement of that probe to find the first fragment in one of the two libraries.
complementary
Such a collection of clones containing contiguous, overlapping pieces of chromosomal DNA is known as a _______.
contig
For large eukaryotic genomes, it is easier to create ________ if the vector can accept very large pieces of chromosomal DNA; artificial chromosomes are such vectors.
contigs
A physical map of a chromosome is constructed by creating a _________ series of clones that span a chromosome.
contiguous
In a large pedigree, it is possible to identify cases where linked micro satellites have segregated due to _______ ____. The frequency of crossing over provides a measure of the map distance between different micro satellites on the same chromosome.
crossing over
By following the transmission of many polymorphic markers within large family pedigrees, it may be possible to determine that particular markers are found in people who carry specific _____-_______ alleles.
disease causing
If the probe is fluorescently labeled, the procedure is called _____ ___ ____ _________ (FISH).
fluoresceence in situ hybridization (FISH)
An RFLP map describes the locations of many different RFLPs throughout the ______. A genetic map composed of RFLP markers is called an RFLP map, and RFLP maps can be used to locate functional genes within the genome.
genome
In the late 1990s, when researcher first began sequencing larger genomes from eukaryotic species, they took the approach of _______ _____ _______.
hierarchical shotgun sequencing
The ability to rapidly sequence large amounts of DNA is often referred to as ___-_____ sequencing.
high-throughput
If the RFLPs are linked, then we see a significantly ______ number of parental types of offspring.
higher
Those that are not linked will ________ sort from generation to generation.
independently
The ___ side describes the locations of RFLP markers.
left
______ ________ also uses molecular markers called micro satellites.
linkage mapping
If the RFLPs were not _____ there would be a 1:1:1:1 ratio of the four types among the offspring.
linked
In linkage studies, the goal is to follow the transmission of many different micro satellites to determine which are ____ linked along the chromosome and which are not.
linked
Those that are ______ tend to be transmitted together to the same offspring.
linked
Mapping refers to the experimental process of determining the relative _____ of genes or other segments of DNA along individual chromosomes.
location
A _______ ______ is a segment of DNA that is found at a specific site along a chromosome and has properties that enable it to be uniquely recognized using molecular tools such as PCR and gel electrophoresis.
molecular marker
Regions of DNA that do not encode genes can be used as _____ ______.
molecular markers
Newer high-throughput platforms are based on different methods of sequencing DNA, referred to as ____-______ sequencing technologies.
next-generation
Restriction enzymes, which recognize _____, can be used to digest a chromosome into many small pieces of different lengths depending upon the spacing of the palindromes; these are known as _______ ________.
palindromes restriction fragments
The cloning of a gene based on its mapped position along a chromosome is called ______ ________.
positional cloning
The process of chromosome walking is frequently used for ______ _______.
positional cloning.
DNA of a cloned gene is used as a _____ to determine the location of that gene.
probe
_____ ______ can occur in palindromes as well as in coding genes, and these mutations can either create or destroy palindromes.
random mutation
The map distance between 4,500/6,500 and the 2,000/1,500 sites is calculated by dividing the number of _______ offspring by the total number and multiplying by 100, which gives us a map distance of 16 mu (map units).
recombinant
Molecular markers serve as ______ _____ along genetic maps; they can be used to determine the approximate location of an unknown gene that causes a human disease.
reference points
The _____ side describes the map distances in map units.
right
Microsatellites are also called ______ ________ _______.
short tandem repeats
Third-generation sequencing platforms read ______ DNA molecules.
single
_____ __________ can be used to distinguish the polymorphism that are specific to the gene of interest.
southern blotting
In fluorescence in situ hybridization, usually ___ spots are usually seen at each site because the probe binds to both sister chromatids.
two
In more recent years, researchers more commonly followed a strategy called _____ ______ _______ sequencing, which bypassed the physical mapping step used in the hierarchical approach.
whole-genome shotgun
