Ch 20 Genomics I: analysis of DNA Flashcards
Genome
total genetic composition of organism or species
Genomics
molecular analysis of an entire genome of a species
Functional Genomics
examining genes to discover how thye interact to produce traits of an organism
Structural Genomics
looking at proteins to see how genes interact to produce traits of an organism
Proteomics
analysis of proteins of an entire genome
Mapping
figuring out relative location of genes or segments of DNA on a chromosome
Three ways to determine the organization of a genome?
Cytogenic Mapping, PHysical mapping, linkage mapping
Cytogenetic Mapping
Determines location of gene along intact chromsome
In what instance is cytogenetic mapping used?
With eukyotes because they have large chromsomes that can be looked at microscopically and individually categorized based on banding pattern of chromsomeos
How accurate is cytogenetic mapping
Not super accurate becase it is only as good as the resolution of the microscope. Limit of approx. 5 mil base pairs. This is like a preliminary step for locating genes before physical mapping is used.
FISH
Flourescence in situ hybridization
-Used to detect location of gene on chromsomes. that is HELD IN PLACE.
How does FISH work?
1) Treat chromsomes of interest with agent that makes them swell and FIX to slide
2) Denature chromsomes
3) Add SINGLE STRANDED dna probes that have biotin
4) Add floresently labeled avidin that bind to biotin
5) View with microscope
* USually have two spots at each location because bound to both chromatids!
Molecular Markers
Small segements of DNA that have unique qualities that can be seen under microscope or recognized by PCR and gel electrophoreses. Serve as guidlines along chromsomes to locate relative position of other genes
Polymorphic Molecular Markers
Used in analysis of pedigrees to find mutant genes causing diseases
How are restriciton enzymes used in physical mapping?
Used to create restriction fragments. Recognize palindromes and break chromsome into many smal pieces depending on spacing of palindromes
How does random mutation effect palindromes?
In can either create or destroy palindromes
EcoRi
Example of a restriction enzyme that recognizes 5-GAATC and digestes the chromosome into small pieces respectivly
RFLP
Restriction fragment length polymorphism. Looking at chromsomes of different individuals in a pop. and seeing different lengths of restriction fragments due to loss or gain of palindromes
Sourthern Blotting uses
used to deterime polymorphism fragments
Microsatellites
Also reffered to as STR’s (Short tandem repeats) are short repetitive sequences interspersed throughout the genome
Most common microsatellite in humans?
CAn (n is anywhere from 5-50 bp)
*found in every 10,000 base pairs
What is sequece Tagged Site and how is it made?
Amplified microsatellite
*amplified using PCR
How are STS sites made in detail?
STS is made using PCR.
1) add PCR primers that specifically target a sequence of a particular chromsome.
2) Many cycles of PCR produce the DNA fragment between the two primers.
3) Use gel electrophoresis that shows the microsatellites
What are the two types of STS instances?
In homozygous individuals two microsatellites will be produced that are identical in length.
In Heterozygous the microsatellites might differ in length
Physical mapping
requires cloning of several peices of DNA and presence of contigious series that overlap in genes
How are chromosomal fragment categorized in physical mapping?
Based on length (bp) and the genes that are contained in said fragment
Contig
a collection of clones containing overlapping pieces of DNA
YAC, BAC, PAC
used to clone large segments of DNA used for eukaryotes
How large of chrosomes can YAC take?
several hundred thousand to 2 million base pairs.
What were BACS and PACS developed from and how many bases can they handle?
developed from F factors and P1 bacteriophage
Can handle 300,000 base pairs
What is a cosmid?
A hybrid between a plasmid vector and a phage lambda. They except smaller pieces of DNA
Positional Cloning
Cloning of gene based on its position along the chromosome
Chromosome Walking
Used for positional cloning.
High throughput sequencing
Sequencing DNA very rapidly
Next Generation Sequencing Technology
New high throughput sequencing methods
Second Generation
uses PCR to amplify large regions of DNA
Third Generation
read single DNA molecules
Pyrosequencing
An example of next generation sequencing
How does Pyrosequencing work?
1) isolate DNA and break into fragment
2) Covalently attach oligonucleiotide adaptors to the 5 and 3’ ends
3) denature DNA into SINGLE STRANDS and attach to bead via the ADAPTORS
4) Emulsify beads so only ONE BEAD PER DROPLET
5) Beads put in picotiter plate. ONE BEAD PER WELL
6) Add sequencing reagents (DNA polymerase, primers, ATP sulfurylase, luciferase, apyrase, adenosine 5 posphosulfate and luciferin)
7) Flow A or T or G or C into each well.
8) When that base adds, a d light will appear
Sequencing by Synthesis
involves identification of each nucleotide immediatly after incorporation ointo a DNA strand by DNA poly
Comparitive Genomics
uses information from genome project to understand the genetic variation amond diff. pops.
