Week 8 Flashcards
what methods are used for nucleic acid detection
in situ hybridisation
gel electrophoresis
blotting
what is in situ hybridisation
hybridisation of a DNA or RNA probe to intact tissue
locates its complementary strand by autoradiography
used to detect mRNA or DNA
what is a dna or rna probe
stretches of single-stranded DNA
used to detect the presence of complementary nucleic acid sequences
what is autoradiography
imaging technique that uses radioactive sources contained within the exposed sample
FISH
florescence in situ hybridisation
used to identify specific sequences in whole tissue with mRNA probes
probes cannot be seen so labelling is needed
gel electrophoresis
separates dna fragments by size
uses an electric current - causes dna to move to the positive charge
how is dna visualised in gel electrophoresis
uses a visible intercalating molecule that binds in between base pairs - gives dna a colour
alternative - radioactive labelling
how is the size of a fragment measured in gel electrophorsis
compared with a fragment of known size
what is blotting
hybridisation technique that allows detection of mrnas and dnas
what is southern blotting
involves transfer of dna from a gel to a membrane
followed by detection of specific sequences by hybridisation
with a labelled probe
what are the three types of dna sequencing
sanger sequencing, next gen. sequencing, PCR
what is sanger sequencing
cloned dna are rapidly sequenced by dideoxy chain termination
uses dideoxynucleoties triphosphare (ddATP) to block further growth - terminates dna synthesis
eventually there will be a mixture of preamture daughter fragments ending at every ddNTP - N is replaces with each ATGC
limitations of sanger
difficult to parallise
cloning is time consuming
large amounts of dna are needed
advantages of sanger
can be done in a single test tube
multiple primers can be used to see overlapping parts of the genome
modern - reusable, can be used for multiple samples
-> cheaper
what is a primer
a single stranded nucleic acid molecule
with 3’ OH to initiate dna pol replication of a paired template
what is integration in next gen sequencing
the efficiency of sequencing pipeline
ssDNA attaches to silicon plate
covered with probes in DNA pol, primer
DNA pol adds one nucleotide at a time as they are all terminating nucleotides
sequence can then be analysed
advantages of Next gen sequencing
no dna library needed - in sanger it is one sequence per tube
in next gen - many sequences are done at the same time by finding the first nucleotide for all sequences then the second and so on.
complementary sequences in overlapping regions can be analysed
adaptor sequences
fragments are modified
added to dna fragments in next gen sequencing
used to unwind dna and create ssDNA - adaptor put on silicon like a chip -
primer to adaptor is added
dna pol with all terminating nucleotides used
what is pcr
allows for amplification of a desired sequence
using primers that anneal to sequence of interest
what is rt pcr
reverse transcriptase pcr
uses reverse transcriptase to convert rna to dna
what is real time or quantitative pcr
detects the products of pcr amplification during synthesis
+more sensitive
what is thermostable dna polymerases
dna pol that can withstand multiple cycles of template denaturation
thermocycler used to amplify dna
dna pol needed to create a copy - two primers on opp sides
northern blotting
hybridisation technique
that allows detection of mRNAs
needs transfer of rna frrom a gel to a membraen
followed by detection of specific sequences with a labelled probe
what are dna microarrays
used to detect levels of all expressed genes in a sample
dna microarrays have known dna sequences on a small chip
used to determine if a sequence has a mutation like in BRCA
what is genome wide transcription analysis
performed using labelled cDNA from experimental samples
hybridised to a microarray containing sequences from all ORFs of the organism
competitive hybridisation
red and green cDNAs to the microarray is proportional to the abundance of each mRNA in the two samples
what are expression vectors
used to introduce a gene into a target cell
contain promoters and ribosome binding sites
- shine delgano sequence
allows for efficient transcription and translation of nay cloned dna
most vectors use the lac operon promoter
transcription from the expression vector is induced by addition of a lactose analogue
what is the shine delgano sequence
enables initiation of protein synthesis by aligning the ribosome with the start codon
SDS gel electrophoresis
proteins are separated accroding to size
SDS is a negatively charged detergent - gives all proteins a negative charge
amount of SDS molecules a protein can bind to is proportional to its size
specific proteins can be detected by blotting with antibodies
western blot
proteins separated by size on SDS gel
transferred to nitrocellulose membrane
detected using an antibody with is coupeld to a fluorophore
nitrocellulose membrane
a matrix
used for high binding affinity to proteins
