Genetics > Chapter 8 > Flashcards

Chapter 8 Flashcards

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1
Q

Genomics

A
  • obtaining+analyzing sequences of complete genome

- Human Genome project -> 3bill$ , 1990-2003

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2
Q

Comparative genomics vs. Fucntional genomics

A
  • compare dif genomes==evolution
    vs
  • when+how gene is used
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3
Q

How to create a physical Map of Genome

A
  • construct genomic library (1+ copy of every DNA sequence in library)
  • make clones= via cloning vectors
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4
Q

cloning vector

A
  • artificially construct DNA Molec

- self replicate in host cell

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5
Q

DNA cloning procedure

A
  1. isolate dna from original
  2. cut dna into pieces
    - > restriction enzymes
  3. insert each piece into cloinign vector (recombination DNA molecule)
  4. Transfer rec. DNA mol into a host cell
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6
Q

Enzyme restriction/endonucleases

A
  • recognize palindromic sequence
  • creates a restriction site
  • base reconistion : 6, 4- base cutters
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7
Q

Types if restriction sites

A
  • blunt * stick less easier then overhang*
  • 5’ overhang
  • 3’overhang
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8
Q

Plasmid Cloning Vectors

A
  1. ori site
  2. selective markers
  3. multi cloning site (MCS or polylinker)
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9
Q

DNA Cloning

A
  • Insert DNA piece into Plasmid Cloning Vectors
  • -> results in recomb dna mole
  • -> ~15kb E.Coli, ~45kb cosmid, ~300kb to 2Mb artifical chromos
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10
Q

Types of Artificial Chromosomes

A

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

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11
Q

Considerations when building Genomic Library

A
  1. size of insert
  2. variation of restriction sites
  3. need OVERLAPPING sequence

*** the need of partial digest

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12
Q

Genomic DNA Fragments

A
  • sorted (size + purified) prior to cloning into vector

- agarose gel electrophresis

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13
Q

Final Steps of creating Genomic Library

A
  • sorted DNA cloned into vector

- inserted/ TRANSFORMED into org/vector

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14
Q

How to have complete coverage in genome Lib

A
  • having millions of clones
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15
Q

Chromosome libraries

A

-seperate based on ind chromo–> cell sorting based on size and shape

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16
Q

DNA sequencing

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A
  • 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
17
Q

Sanger+ Dideoxy sequ

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A

dideoxynucleatodide (ddNTP) Dna precussor

–> 3’c has H+

18
Q

Radioactive vs. Automated method

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A
  • 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
19
Q

Pyrosequencing==> pyrograms

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A
  • 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
20
Q

Assembling

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A

-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

21
Q

Annotation of variation

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A
  • single nucleotide polymorphesins==detailed map
  • -> useful as DNA marker
  • -> 1 SNP per 1000 bp
  • -> nearby SNP groups can be inhertated (haplotype)
22
Q

Haplotype

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A
  • isolate gene associated w/ disease

- understand inheritance of complex traits

23
Q

ID and annotation gene sequence using cDNA

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A
  • generating a cDNA
  • double strand cDNA is cloned into vector
  • build cDNA library (“linkers” are used for restriction site)
24
Q

how to generate cDNA

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A
  1. anneal an oligo (dT) primer to mRNA via poly tail
  2. add reverse transcriptase retrovirus= cDNa (mRNA hybrid)
  3. remove mRNA by Rmase H /nuclase= cleaves
  4. Degrade mRNA = primers –> DNA pol synthesis new strand w/dNTP
  5. DNA pol removes RNA primers +ligase fills in gap
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