DNA and genome sequencing Flashcards
what is DNA sequencing
figuring out the order of DNA nucleotides or bases in a piece of DNA
what is high resolution electrophoresis
motion of dispersed particles relative to a fluid under the influence of a uniform electric field
what can gel electrophoresis be used for
can be used to separate fragments of DNA
what is methyl interference assay
analytic method to identify nucleotides that are important for DNA binding
what is a primer (oligonucleotide)
strand of nucleic acid that serves as a starting point for DNA synthesis
what is a Klenow fragment
large protein fragment produced when DNA polymerase I is cut by the protease subtilisin enzyme
what is polyacrilamide gel electrophoresis (PAGE)
technique used to separate DNA or protein molecules, its high resolution allows the separation of a single-base pair of DNA
what is autoradiography
an image on an X-ray film produced by the pattern of decay emissions of a radioactive substance
what is genome sequencing
determination of the order of the bases in the DNA of a genome
what are non-protein coding sequences
all the sequences in a genome that do not contribute to make a protein, including regulatory regions and pseudogenes
what is a genome map
set of landmarks in a genome
what are the two procedures involved in DNA sequencing
- selective termination of DNA synthesis at specific nucleotides (A, C, G or T)
- high resolution electrophoresis
what are examples of sequencing methods
Maxam and Gilbert method
Sanger dideoxy method
how does the DNA strand grow and what is produced
new nucleotides added
three phosphate, two removed, one phosphate bind to carbon 3 of sugar form a molecule of water
what is the Maxam-Gilbert method for sequencing a DNA molecule - functions
- based on chemical modification of DNA and subsequent cleavage at specific bases
- it is technically complex
- it uses hazardous chemicals
- it is difficult to scale it up
what HAPPENS in Maxam-Gilbert method for sequencing a DNA molecule
DNA molecule with radioactive label – phosphorus 32 is radioactive
Different tubes, each will destroy different bases in the DNA
Kinase reaction using phosphate
Cut DNA using hot piperidine
Add new substances to the test tubes, these will start cutting the DNA segments
what method led to the methylation interference assay
fragments in the four reactions are electrophoresed in a denaturing acrylamide gel for size separation, gel is exposed to an X-ray film for autoradiography
what is the methylation interference assay used for
to map DNA binding sites for DNA binding proteins
why is the Sanger dideoxy method for sequencing a DNA molecule good
more efficient and uses fewer toxic chemicals and lower amounts of radioactivity
what does the Sanger dideoxy method require
defined primer that binds to the 3’ end of the template strand to be sequenced
what is the Sanger dideoxy method based on the use of
dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators
what must the primer of the Sanger dideoxy method for sequencing a DNA molecule have
must be tagged at its 5’end with a radioactive 32P-phosphate (gel) or a fluorescent dye (automated sequencer reading)
how is the DNA synthesised and how is it terminated in the Sanger dideoxy method for sequencing a DNA molecule
synthesis of a new strand of DNA is initiated from the primer, and a chain termination process is used to stop synthesis selectively at any one of the four DNA nucleotides
how does the DNA synthesis occur in Sanger dideoxy method for sequencing a DNA molecule
incorporation of a 2’-, 3’-dideoxynucleotide, which leaves no 3’-OH group for further chain growth
what does the Sanger dideoxy method for synthesising a DNA segment produce
series of fragments with various lengths will be generated which can then be used to read the nucleotide sequence
what is do the four identical reaction mixtures require in the Sanger dideoxy method in the lab
Same template – DNA want to sequence
Labeled primer
DNA polymerase
Deoxyribosenucleoside triphosphates: dATP, dTTP, dCTP, dGTP.
One of the four nucleotides in dideoxy (dd) form: ddATP, ddTTP, ddCTP, ddGTP
what is needed to support the 5’ to 3’ DNA synthesis in the Sanger dideoxy method in the lab
Klenow fragment of DNA polymerase I
when does synthesis of new strand stop
Synthesis of the new strands continues until a dideoxyribonucleotide is incorporated
what is used to detect the radioactive bands
autoradiography
sequence of the original template strand can be deduced from the bands on the autoradiograph
what lowers the cost of DNA sequencing
High-throughput sequencing technologies
examples of high-throughput sequencing technologies
illumina (solexa) sequencing, DNA nanoball sequencing
what do we need to know about the genome
- how the genome is organised
- structure of genes
- function of genes
- how these genes are related
- how various parts of the genome are coordinated
- genes account for less than 25% of the DNA in the human genome
- unidentified secrets of life
how do you sequence a genome
break the genome into pieces either using 1. clone-by-clone approach 2. whole-genome shotgun sequence the pieces reassemble them in proper order
what do the non-protein coding sequences make up in prokaryotes and eukaryotes
prokaryotes - only a small fraction of the DNA of prokaryotes
eukaryotes - as their complexity increases, so too does the proportion of their DNA that does not code for protein
what does the genome map include
sort DNA sequences
regulatory sites
promoters
genes
what is a genome map like
one-dimensional-it is linear; it looks like a straight line with landmarks noted at irregular intervals along it
what are the landmarks in a genome map
combinations of letters and numbers that stand for genes or other features
what is the use of a genome map
need map to understand the genome sequence
can help in the sequencing of a genome
help scientists study genes
if us clone-by-clone method what is the map like
indispensable to determine where each clone belongs in the genome
what are genome maps used for
position where a clone came from
they help to find other important parts of the genome
they help to keep track of which part of genomes are studied by different groups
they help to identify clusters of related genes and gene-rich regions of a genome
they can be used to compare genomes of different species and help to understand the process of evolution
