Whole genome shotgun sequence
obtaining and assembling the seq of a genome . determining the seq of several DNA segments generate by breaking long chromosome into short chromosome
traditional WGS
relied on clones in micro cell. used the sanger dideoxy seq. start with construction of genome library. short dna segment inserted into chromosome to form vector.
Next generation WGS
cell free. millions of fragments isolated and seq parallel. advanced fluid tech. high throughput.
pyrosequencing
dna seq technology that is based on the generation and detection of a pyrophosphate group liberated from a nucleotide triphosphate. example of a next generation.
1. template library made of ssDNA
2. amplified into copies using PCR
3. each bead put to wall
bioinformatics
analysis of entire genomes. contain info on numbers and types of genes, gene products and number, DNA and RNA binding sites.
comparative genetics
closely and distinctly related species for evolutionary insight uses conserved seq as a guide to study gene function of the relation of genome sequences of two or more species
1. decide which genomics to compare. (phyology)
2. identification of closely re
orthologs
gene in different species that evolved from a common ancesteral gene by speciation
paralogs
genes that are related by gene duplication in genome
functional genomics
use of expanding variety of methods like reverse and forward genetics to understand gene function and to delineate networks of interacting genes
reverse genetics
disrupt function of a specific gene starts with known cloned dna mrna and protein and tries to dirupt the molecule to assess the role of normal gene product .
target mutagenesis (most) create phenocopies
introduce random mutation
create phenocopies
forward genetics
starts with analysis to identify a set of genes as candidates for encoding the bio property of interest then induces mutants targeted specifically to those genes and then examines the mutant phenotypes to see if they affect the property under study
steps in obtaining a genomic seq.
1. cut many genomic copies into random fragments
2. sequence each fragment
3. overlap sequence reads
4. overlap contigs for complete sequence
contigs
overlapping reads assembled into units contagious of touching group of overlapping cloned segments
scaffolds
supercontig. collection of joined together contigs in which there may be unsequenced gaps connected by paired seq reads
EST
short cdna seq reads only the 5 or 3 ends or both are sequenced
-cdna and ets do not align= intron
examples of binding sites for proteins
1. regulatory proteins binds dna to transcription regulatory elements
2. rna pol binds to promoter
3. operator (dna seq and repressor protein)
RNAi technique
powerful method for inactivating specific genes with only knowing the genomic sequence. double stranded rna is made with sequences homologous to part of the gene under study and is introduced into a cell. risc then degrades native mRNA that is complementa
yeast two hybrid system
protein to protein interaction. physical interaction btw proteins basis of test is transcription activator encode by yeast GAL4 gene uses bait protein and target protein to restore function of GAl4 protein that activates a reporter gene Lacz
CHIP technique
studies the protein to dna interaction isolates the dna and its associated proteins in a specific region of chromatin
1. cross link proteins to dna
2. break chromatic
3. add antibody to target protein/purify
4. reverse cross links to separate DNA protein
microarrays can detect diff in genes expression
1. extraction of mrna from cells or tissues
2. synthesis of florescent dye labeled cdna probes
3. hybridization
4. detection of signal
5. image analysis
3 ways to disrupt gene function
DsRNA-> into cell
trangene with reverse repeat is introduced into genome -> RNA transcript forms a self comp stem and loop
transgene containing two promoters in opp orientations to genome-> RNA transcribed
transcriptome:
seq and expression patterns of all transcripts
proteome
seq and expression patterns of all proteins
interactome
complete set of physical interactions between proteins and dna segments
What approach would you use to sequence the genome of a newly discovered bacterial species?
A.Microarrays
B.Whole genome shotgun approach
C.Ordered-clone sequencing
D.Expressed sequence tags (ESTs)
B.Whole genome shotgun approach
A segment of cloned DNA containing a protein-encoding gene is radioactively labeled and used in an in situ hybridization. Radioactivity was observed over five regions on different chromosomes. How is this result possible?
A.The clone may contain DNA that
D.All of the above are correct.
In an in situ hybridization experiment, a certain clone bonded to only the X chromosome in a boy with no disease symptoms. However, in a boy with Duchenne muscular dystrophy (X-linked recessive disease), it bonded to the X chromosome and to an autosome. C
C.Yes, because the clone hybridizes to a translocation breakpoint between the X and autosome.
In a genomic analysis looking for a specific disease gene, one candidate gene was found to have a single-base-pair substitution resulting in a nonsynonymous amino acid change. What would you have to check before concluding that you had identified the dise
all (check before conclusion)
To inactivate a gene by RNAi, what information do you need?
A.Gene position
B.Sequence of the gene
C.Promoter sequence
D.All of the above
B.Sequence of the gene
Prokaryotes transposable elements
IS: short sequences elements that can move themselves to new positions but don't carry genes other than those needed for their movement can move on same or different chromosomes
transposons: longer seq that carry genes for movements and other genes for mo
euk transposable elements
class 1 retrotransposons
class 2 dna transposons
retrovirus
ssrna virus that employs a double stranded dna intermediate for replication have env gene
ltr
direct repeat of DNA seq at the 5 and 3 seq both contain the gened gag and pol and use revers transcriptase
target site duplication
short direct repeat dna sequence adjecent to the ends of a trnasposable element that was generated during the elements integration into the host chromosome
replicative
prokaryotes a copy of the element into a target site leaving one copy behind at the original site
conservative
prokaryotes. direct excision of the element and its reinsertion into a new site. no replication. element is excised from the chromosome or plasmid and is integrated into new site
p element
short inverted repeats that encodes a single protein
Which of the following best describes why plasmids can gain multiple drug-resistant genes as they mix and spread through different strains of cells?
A. The genes are duplicated and mutated during the normal replication process.
B. Bacteria have a single s
C. The genes are flanked by inverted repeats, a unit known as a transposon, which allows them to jump between DNA molecules.
How do the properties of P elements in Drosophila make gene-transfer experiments possible in this organism?
A. They can be used to generate new mutations by random insertion and gene knockout.
B. They can be used to insert almost any gene into the Drosoph
D. All of the above are correct.
flies
The insertion of transposable elements into genes can alter the normal pattern of expression. In the following situation, what is the possible consequence on gene expression?
A Ds element that was inserted into the middle of an intron excises imperfectly
A. The excision of Ds will likely have no effect on intron splicing.
In browsing through you find 3 homologs in the chimp two in humsn. what are the alt? and how can you distinguish them?
one could have been lost after the human ancestors diverged from the chimp or the common ancestor had two and a duplication has occurred in the chimp line
one would need to determine how many copies are present in other great apes (evolution)