Flashcards in Topic 4 - chapter 10 Deck (38)
What does gel electrophoresis do?
separates DNA fragments of different sizes when voltage is applied
Is DNA +vely or -vely charged?
What is hybridisation?
a sensitive way to detect specific nucleotide sequences based on complementary basepairing
How does hybridisation basically work?
DNA double helices + hight temp/high pH -> denaturation to single strands (H bonds break) + slowly cool/low pH -> renaturation restores DNA double helices
Which two techniques does Southern blotting combine?
electrophoresis & complementary basepairing
What are DNA probes used for?
to recognise a desired nucleotide sequence
Can recombinant DNA be copied inside bacterial cells?
What can be used to clone DNA?
Specialised plasmid vectors
cDNA libraries represent what?
mRNA produced by a particular tissue or organ
What is PCR?
Polymerase chain reaction - used to amplify selected DNA sequences
are circular extrachromosomal pieces of DNA with an origin of replication and do NOT contain essential genes
A genomic library contains...
all DNA from an organism packaged into small fragments cloned into plasmids
What are positive clones and how can they be found?
positive clones are desired clones containing part or the whole gene of interest and can be found via hybridisation
What is a cDNA library? What does it contain?
a representation of the mRNA produced by a certain tissue
contains only expressed sequences - so <1.5% of the human genome that are coding sequences
From tissue (e.g brain) to cDNA copy...
1. Tissue cells (brain) 2. lyse & purify mRNA 3. mRNA with poly A tail at 3' end 4. hybridise with poly T primer 5. make DNA copy with reverse transcriptase 6. degrade RNA with RNase H 7. synthesise complimentary DNA strand using DNA polymerase with 5' end of original mRNA acting as primer 8. double-stranded cDNA copy of original mRNA
Describe the PCR aloud...
slides 30 - 32...
What are some applications of PCR?
- amplifying DNA strands
- 1st step in cloning process (genomic/DNA clones or cDNA/mRNA)
- very sensitive detection system/diagnostic method (HIV eg)
- very sensitive identification method with extremely high resolution (short tandem repeats (STR) locus in a single individual - maternal & paternal/paternity tests with electrophoresis)
Studying gene function usually involves what?
constructing recombinant DNA molecules with plasmid vectors
Understand the process of sequencing...
slides 38 - 40
What are the 2 automated sequencing approaches used in human genomes?
- 'clone by clone'
What is an 'expression vector'? What are it's implications?
Specialised vectors used in DNA cloning that contain appropriate transcription & translation signals so that inserted gene is expressed at very high levels
What is 'shotgun' sequencing? What are its drawbacks?
sequencing (determining the order of nucleotides) method where genome is broken into smaller, overlapping fragments then sequenced and genome assembled based on overlapping sequences
Drawbacks - only good for sequencing small genomes
What is clone-by-clone sequencing?
technique where the human genome is broken into 100-200kb fragments -> into BAC -> E. coli -> divide (with BACs) -> many overlapping cloned fragments -> use restriction enzyme to generate 'signature' of each clone -> use shotgun method to determine nucleotide sequence of each BAC seperately -> assemble whole genome by stitching together the sequences of 1000s of BACs
What is the 'eternal' pathway?
Recombinant DNA technique that begins with a protein of unknown function -> isolate gene encoding it -> churn out ++ protein for study of structure & activity
What are 2 common reporter proteins?
enzymes beta-galactosidase & green fluorescent protein (GFP)
What are reporter genes used for?
to monitor when & where a gene is expressed
What is & in situ hybridisation used for? Probes?
to monitor the time & place mRNA product of a gene is expressed (good if gene's final product is RNA rather than protein)
Probes include nucleic acid labeled with fluorescent dyes or radioactive isotopes
Some applications of in situ hybridisation method...
- detect the presence of a virus in the cell eg. human papillomavirus (HPV)
- embryonic development
- locate genes on chromosomes (chromosome 5)
What is FISH?
Fluorescence in situ hybridisation