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Basic Laboratory Techniques

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anneal with designed primers. synthesize DNA. DNA polymerase ... anneal into double strand. 65 68 oC, in plastic bag or chamber. Southern (Northern) Blotting ... – PowerPoint PPT presentation

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Title: Basic Laboratory Techniques


1
Basic Laboratory Techniques
  • Microarray Database Systems
  • 9/23/2002

2
Outline
  • Basic genetic engineering tools
  • labeling, gel electrophoresis, hybridization
  • DNA sequencing
  • DNA cutting
  • DNA amplification
  • DNA cloning
  • PCR

3
Why Details?
  • to study papers
  • to understand and communicate with biologists
  • to record important procedures that affect the
    data in databases or analysis tools
  • to integrate data coming from different domains

4
Isolation of DNA and RNA
  • open cells in the sample
  • cell disruption remove cell wall, membrane
  • as gentle as possible
  • separation of nucleic acids from others
  • deproteinisation
  • purification recovery of nucleic acids
  • DNA RNase to digest RNA
  • RNA oligo(dT)-cellulose to bind poly(A) tails of
    eukaryotic mRNAs

5
Syntheis of cDNA
mRNA
AAAAAA 3
  • prepare mRNA
  • 3 poly(A) tail
  • insert a primer
  • in case of full length synthesis, oligo(dT)
    matching 3-end poly(A) tail of the mRNA
  • reverse transcription
  • all four dNTPs supplied (can be labeled)
  • DNA polymerases

AAAAAA 3
HO - TTTTTT 5
AAAAAA 3
TTTTTT 5
cDNA
TTTTTT 5
OH
TTTTTT 5
double-strand cDNA
6
Nucleic Acid Labeling
  • End Labeling
  • adding a modified, or radioactive, nucleotide to
    one end of a DNA fragment
  • 5-End labeling add a radioactive phosphate
  • 3-End labeling add one or more nucleotides

7
Nucleic Acid Labeling
  • By Primer Extension
  • denature DNA
  • anneal with designed primers
  • synthesize DNA
  • DNA polymerase
  • incorporating labeled nucleic acids

5
3
5
3
HO
Primer
5
3
Primer
DNA polymerase I
8
Non-radioactive Labeling
  • safer, faster
  • fluorescent or non-fluorescent

9
Gel Electrophoresis
  • label molecules
  • with radioactive isotopes or fluorescent dyes
  • charge the molecules and put them in one end of a
    gel medium with strong electric field
  • the molecules move in the gel with speed
    inversely proportional to their size
  • logarithmic relationship
  • measure the relative weight through the relative
    offset

10
Genome Sequencing
  • Chromosome
  • a long strand of DNA (molecule)
  • Locus (loci)
  • the location of a gene in a chromosome
  • Physical Map
  • the exact location, in base pairs, of a gene in
    a chromosome

11
Sequencing Top-down
  • Prepare 4 groups of DNAs
  • Break the DNAs into pieces end with A, T, G, C,
    respectively
  • Maxam-Gilbert (chemical)
  • Sanger-Coulson (enzymatic)
  • Run the 4 DNA groups using gel electrophoresis in
    4 lines
  • Read exact sequence
  • up to 700 bps long

12
Automated DNA Sequencing
  • four different, fluorescent dye-labeled chain
    terminators
  • one tube
  • one gel lane
  • read by laser
  • automated sample processing
  • high throughput

13
Sequencing Shotgun Method
  • Break DNAs into pieces randomly
  • Select a subset for further processing
  • amplification
  • Sequence the pieces
  • Paste the pieces together
  • computer

14
DNA Cutting
  • Restriction Enzyme
  • Proteins that cut DNA at specific points called
    restriction sites
  • determined by the local base sequences
  • may leaving sticky ends
  • Example EcoRI looks for GAATTC
  • GAATTC gt G AATTC
  • CCAGAATTCTCG gt CCA GAATTCTCG
  • GGTCTTAAGAGC GGTCTTAA GAGC

15
DNA Cloning
  • Generate an initial DNA fragment
  • Join to a vector (DNA cutting involved)
  • Introduce into a host cell and let the cell
    multiply
  • extract the inserts
  • recombinant refers to DNA produced this way

16
Popular Vectors
  • Plasmids
  • circular DNA exists in bacteria
  • afford up to 15 kbp inserts
  • Phages
  • viruses that infects bacteria
  • for inserts up to 25 kbp (limited by capsule)
  • YAC (yeast artificial chromosome)
  • extra, artificially made chromosome to yeast
  • for large inserts (a million bps)

17
Libraries
  • Library A collection of independent clones
  • Genomic Library a set of clones representing the
    entire genome of an organism
  • represent engire genome as a set of overlapping
    cloned fragments
  • randomly
  • maintained in stable form with no
    misrepresentation of sequences
  • cDNA Library
  • from mRNA

18
Polymerase Chain Reaction
  • Iterations of phases
  • denature double-strand DNA
  • heat
  • convert single strands into double strands
  • need primer
  • Taq thermostable polymerase

5
3
5
3
5
3
primer
primer
3
5
Taq
5
3
primer
primer
3
5
Taq
8
16
19
Probe and Target
  • Probes
  • oligonucleotides or clones (DNA, RNA) with known
    sequences
  • Targets
  • biomaterials under study

20
Hybridization
  • Joining together (artificially) separated nucleic
    acid strands
  • denature probes and/or targets
  • into single strands, by heat (90 oC)
  • either one can be immobilized
  • anneal into double strand
  • 65 68 oC, in plastic bag or chamber

21
Southern (Northern) Blotting
  • Run targets DNA (RNA) in gel
  • Denature DNA into single strands (Northern)
  • Transfer from gel into membrane
  • immobilize targets
  • Hybridization with labeled probes
  • Wash out non-hybridized materials
  • Read out image data

22
Microarray
  • immobilize massive probes on a chip
  • hybridize with labeled target samples
  • extract relative abundance of the base pairing
  • investigate gene expression

Labeled sample
Hybridization
Scanning
23
Impact of Microarray
  • General
  • massive parallel from gene to genome study
  • functional genomics more than sequence
  • Technical
  • advanced image analysis, noise handling
  • large-scale data storage and management
  • large-scale data processing
  • heterogeneity in technology and data format
  • Long-term
  • full feedback loop from experiment to analysis
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