Chapter 8 Flashcards
Genomics
- obtaining+analyzing sequences of complete genome
- Human Genome project -> 3bill$ , 1990-2003
Comparative genomics vs. Fucntional genomics
- compare dif genomes==evolution
vs - when+how gene is used
How to create a physical Map of Genome
- construct genomic library (1+ copy of every DNA sequence in library)
- make clones= via cloning vectors
cloning vector
- artificially construct DNA Molec
- self replicate in host cell
DNA cloning procedure
- isolate dna from original
- cut dna into pieces
- > restriction enzymes - insert each piece into cloinign vector (recombination DNA molecule)
- Transfer rec. DNA mol into a host cell
Enzyme restriction/endonucleases
- recognize palindromic sequence
- creates a restriction site
- base reconistion : 6, 4- base cutters
Types if restriction sites
- blunt * stick less easier then overhang*
- 5’ overhang
- 3’overhang
Plasmid Cloning Vectors
- ori site
- selective markers
- multi cloning site (MCS or polylinker)
DNA Cloning
- Insert DNA piece into Plasmid Cloning Vectors
- -> results in recomb dna mole
- -> ~15kb E.Coli, ~45kb cosmid, ~300kb to 2Mb artifical chromos
Types of Artificial Chromosomes
a. Bacterial AC (BAC)
1. F Factor (bacteria ORI)
2. Selectable marker
3. MCS
b. Yeast AC (YAC)
1. Telomere (TEL) indicate end of line
2. Centromere (CEN)
3. Selectable markers (TRP1+URA3)
4. Origin of rep sequence (ARS)***eukaryotes only
5. Ori site aswell (allow circular empty vector) *prokaryotes + unique to bacteria prior to insert into yeast
6. MCS
Considerations when building Genomic Library
- size of insert
- variation of restriction sites
- need OVERLAPPING sequence
*** the need of partial digest
Genomic DNA Fragments
- sorted (size + purified) prior to cloning into vector
- agarose gel electrophresis
Final Steps of creating Genomic Library
- sorted DNA cloned into vector
- inserted/ TRANSFORMED into org/vector
How to have complete coverage in genome Lib
- having millions of clones
Chromosome libraries
-seperate based on ind chromo–> cell sorting based on size and shape
DNA sequencing
- smaller 18-20 bp more likely to appear
1. break bonds=single strand 3’ to 5’
2. cool down + add sequence primer
3. supply cofactor +DNA polymerase ===new DNA - Sanger and Dideoxy sequencing
- Radioactive vs. Automated method
- Pyrosequencing==> pyrograms
Sanger+ Dideoxy sequ
dideoxynucleatodide (ddNTP) Dna precussor
–> 3’c has H+
Radioactive vs. Automated method
- label 1 nucleotide per tube
-lower on gel is smallest
-reads 5’ to 3’
vs - tags w/ color that is detected
Or
-Capillary electrophoresis= band reading
Pyrosequencing==> pyrograms
- 4 enzyme system
1. DNA poly: add nucleotide to growing DNA strand
2. ATP sulfurylase: convert PPi( Pyrophosphate) to ATP
3. Luciferase : convert ATP to light
4. Apyrase: remove nuc. that are unbound
Assembling
-shotgun approach :
*genome broken into partiallly overlapping
*library w/ smaller frag ~2kb= sequenced
–> it can lead to missassembling
*library with larger gra=unique sequences
==== higher fold # = better guide
Annotation of variation
- single nucleotide polymorphesins==detailed map
- -> useful as DNA marker
- -> 1 SNP per 1000 bp
- -> nearby SNP groups can be inhertated (haplotype)
Haplotype
- isolate gene associated w/ disease
- understand inheritance of complex traits
ID and annotation gene sequence using cDNA
- generating a cDNA
- double strand cDNA is cloned into vector
- build cDNA library (“linkers” are used for restriction site)
how to generate cDNA
- anneal an oligo (dT) primer to mRNA via poly tail
- add reverse transcriptase retrovirus= cDNa (mRNA hybrid)
- remove mRNA by Rmase H /nuclase= cleaves
- Degrade mRNA = primers –> DNA pol synthesis new strand w/dNTP
- DNA pol removes RNA primers +ligase fills in gap
