L6 Hybridization Flashcards
Using specific probes that are labelled specific sequences of DNA can be identified
Blotting/Hybridization Techniques
Transfer of an RNA sample separated and identified using DNA or RNA probes
Northern Blot
Transfer of a DNA sample separated and identified using DNA or RNA probes
Southern Blot
Transfer of a protein sample separated and identified typically using an antibody
Western Blot
ssDNA that will base pair with a complementary sequence of either RNA or DNA
Annealing/hybridization
Probes (detection with probes)
Stringency: heat, chemical, salt affect binding sensitivity and specificity
Annealing/hybridization
- Radioactive (P32) label
- Chemiluminescent compound
- Fluorescent compound
- Enzymatic label
ID of target detected/identified by labeling probes with
Separated Nucleic Acids are blotted onto _____ ______ by suction of buffer through gel and paper
nitrocellulose paper
Separate DNA fragments by ___ and ___ ________.
RE, gel electrophoresis
ASO (stands for?)
Allele-specific Oligonucleotide
How many probes does ASO have for each allele?
2
Cystic fibrosis - loss of ____________ leads to one form of the disease
phenylalanine 508
Probes
- AsOn = WT (wild type)
- ASOx = mutant cystic fibrosis gene (loss of phenylalanine 508)
ASO for Cystic Fibrosis Detection
RNA:DNA hybrids are captured onto a microtiter well coated with capture antibodies specific for RNA:DNA hybrids
Capture hybrids
- DNA is denatured
- Hybridized to an RNA probe
- Captured by bound anti-DNA/RNA antibodies
Chemiluminescence detection of hybrid molecules (DNA/RNA)
PCR steps
- Denaturation
- Annealing
- Extension (elongation)
Denaturation temperature
94
Extension (elongation) temperature
72
Taq polymerasae
Thermophilus aquaticus
Termination temperature
4
Repeat for ___ - ____ cycles
2 - 30
Taq polymerase isolated from bacterium ________ that lives in ____ ______ and hydrothermal vents
T. aquaticus, hot springs
Taq optimum temp ______ for activity polymerization at a rate of about ____ nucleotides per second per enzyme molecule
75 - 80, 150
produce a DNA copy of the RNA template using either random primers, an oligo(dT) primer or sequence-specific primers
RT-PCR
The _____ ______of the RNA template are crucial to the success of RT-PCR
quality and purity
3 enzymes for RT-PCR
Reverse transcriptase
RNAse H
Taq DNA polymerase
More clinically used because it is isothermal and begins w. RNA
NASBA and TMA Isothermal
Hybrid capture
bDNA
Signal Amplification Methods
Anti-DNA-RNA hybrid antibody
Hybrid capture
More recent efforts have focused on the development of bDNA assays for the quantification
HIV-1 and hepatitis C virus (HCV) RNA,
Sequencing method of choice
Sanger method of choice
Requires ssDNA template, DNA primer, DNA polymerase, labeled nucleotides and modified nucleotides to terminate DNA elongation
Sanger
DNA sample divided into 4 separate reactions (tubes)
Sanger method
______ will prevent addition of further nucleotides
ddNTPs
4 tubes in Sanger
G =
A =
C =
T =
ddGTP
ddATP
ddCTP
ddTTP
Ultra-high throughput sequencing
The whole human genome (as many as 3 billions NA) with (Illumina) may be run in parallel in 30 hours
Next Generation of Sequencing (NGS)
- Fragmentation of DNA
- Adapter are ligated
- Denature to single strands
- Formation of clonal cluster or bead populations
- PCR amplifies DNA strands on flow cell or beads
Basic major procedure of next generation sequencing
powered by semiconductor chips technology
Ion torrent
based on sequencing-by-synthesis operations and reversible dye-terminators
Illumina (SOLEXA) sequencing
- Add dNTPs, and DNA polymerase enzyme to elongate DNA strands
- Repeat until we have millions of dense clusters of DNA. The reverse strands are then cleaved and washed aways
Bridge Amplification - Illumina SBS technology
Use Allylamine-dUTP for
FISH DNA Probe Labeling
Harvest ______ (Will be stopped at metaphase) treated cultured cells in hypotonic solution → full cells – FISH technique
colcemid
Burst cell and load cells onto ____ ______
- FISH technique
glass slide
- Used to identify the presence and location of a region of DNA or RNA (large fragment) within morphologically preserved chromosome preparations, fixed cells or tissue sections
- This means you can view a segment or entire chromosome with your own eyes
- Was often used during M phase but is now used on I phase chromosomes as well
FISH Clinical Utility
P24
Gag
Reverse transcriptase, integrate, protease
Pol
Envelope components; gp120, gp41
Env
