Libraries Flashcards
What design features do primers have?
Tm ~55'C 3' G/C No long complementary repeats 20-30nt long 50% G/C Start/stop codon
What is the cycle of PCR?
94’C denaturisation
55’C annealing
72’C extension
What alternatives can be used for PCR denaturisation?
alkaline denaturisation of DNA
Which polymerases can be used for PCR?
Taq with no exonuclease
Kod/Pfu with 3’-5’ proof reading
How many PCR cycles are required for production of product?
4
How many PCR cycles are used for amplification?
24-30
What is the outline of the cloning protocol?
Purify vector and gene fragment from library
Isolate gene by restriction digestion of PCR
Ligate gene and vector
Transform into colonies
Check recombination by antibiotic resistance or PCR
Extract vector
Transform into expression colony
Isolate protein product
What are libraries?
Collection of fragments of the entire genome contained within a collection of colonies
How is the genome fragmented for libraries?
Partial digestion by high frequency restriction sites (Sau every 250bp) or mechanical
What vectors are used for library production?
lamda phage (20kb) cosmid (40kb)
What are libraries used for?
PCR amplification,
functional/manipulative genomics
How are cDNA libraries produced?
mRNA isolated by oligo(dT) columns for Poly(A) tail
reverse transcriptase produces cDNA
What was the original use of libraries?
Ordered, overlapping libraries for sequencing with radioprobes
What is the modern approach to sequencing of gene fragments from libraries?
Shotgun approach fragments genome and sequences are aligned without ordering
What are the types of sequencing?
Sanger, Illumina, Maxam Gilbert
How is reannealing of DNA prevented during Sanger sequencing?
Heated to denature and high [primer]
Which type of gene sequencing does not require librarys?
Illumina sequencing is continuous so doesn’t require multiple fragments
How does Maxam-Glibert sequencing work?
4 reactions of chemical cleavage: A>G, G>A, C+T, C. Strands radioactively labelled and separated by electrophoresis for sequencing
What are the 2 types of radioactivity of NTPs?
alpha at first phosphate incorporated
beta
gamma at last phosphate released as PPi
Which Maxam-Gilbert reaction is unreliable?
C
Which enzyme adds radioactive end labels to gene fragments?
polynucleotide kinase
Which nucleotide structure has the highest Tm?
RNA:RNA hybrids
How can libraries be screened?
probes to identify target fragments similar to one previously sequenced
What factors cause high stringency probe hybridisation?
T close to Tm, low salt concentrations
What affect does formamide have on Tm?
Lowers it by breaking H bonds
What type of annealing do heterologous probes have?
Low stringency
How can probes be labelled?
radioactive, antibody-enzymatic coupling or fluorescent
What are the methods of labelling radioactive probes?
Single or multiple end labelling, nick translation, random hexamer extension, riboprobes, PCR
Which end of DNA do end labels join?
5’
How are DNA nicks produced?
Polymerase 1 5’-3’ exonuclease
How are end labels added?
y-32P by polynucleotide kinase
How do riboprobes act?
incorporated into a vector under control of a phage polymerase promoter
produce RNA:RNA hybrids that are resistant to ribonucleases
What techniques detect radioactive probes?
autoradiography or phosphorimager
Which enzymes are commonly used to label probes?
Alkaline phosphatase, horseradish per oxidase or luciferase produce visible products
How can probes be detected using antibodies?
Biotin-UTP detected by streptavidin
Digoxigenin derived UTP detected by antibodies
What product does alkaline phosphatase produce?
4-nitrophenyl phosphate
What does horseradish peroxidase produce?
3,3-diaminobenzidine
What do southern blots detect?
DNA
How does a southern blot work?
Electrophoresis Acid degradation of large fragments Alkali denaturisation Fixation to nitrocellulose by UV or heat Non specific prehybridisation Probe hybridisation and detection
What are the differences in a northern blot?
mRNA not DNA
Glyoxal or formaldehyde denaturisation
Where does in situ hybridisation occur?
In dead, fixed tissues
What do microarrays detect?
mRNA or cDNA annealing to immobilised DNA, multiple genes at any time
