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Applied Genetics > DNA and genome sequencing > Flashcards

DNA and genome sequencing Flashcards

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1
Q

what is DNA sequencing

A

figuring out the order of DNA nucleotides or bases in a piece of DNA

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2
Q

what is high resolution electrophoresis

A

motion of dispersed particles relative to a fluid under the influence of a uniform electric field

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3
Q

what can gel electrophoresis be used for

A

can be used to separate fragments of DNA

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4
Q

what is methyl interference assay

A

analytic method to identify nucleotides that are important for DNA binding

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5
Q

what is a primer (oligonucleotide)

A

strand of nucleic acid that serves as a starting point for DNA synthesis

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6
Q

what is a Klenow fragment

A

large protein fragment produced when DNA polymerase I is cut by the protease subtilisin enzyme

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7
Q

what is polyacrilamide gel electrophoresis (PAGE)

A

technique used to separate DNA or protein molecules, its high resolution allows the separation of a single-base pair of DNA

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8
Q

what is autoradiography

A

an image on an X-ray film produced by the pattern of decay emissions of a radioactive substance

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9
Q

what is genome sequencing

A

determination of the order of the bases in the DNA of a genome

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10
Q

what are non-protein coding sequences

A

all the sequences in a genome that do not contribute to make a protein, including regulatory regions and pseudogenes

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11
Q

what is a genome map

A

set of landmarks in a genome

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12
Q

what are the two procedures involved in DNA sequencing

A
  1. selective termination of DNA synthesis at specific nucleotides (A, C, G or T)
  2. high resolution electrophoresis
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13
Q

what are examples of sequencing methods

A

Maxam and Gilbert method

Sanger dideoxy method

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14
Q

how does the DNA strand grow and what is produced

A

new nucleotides added

three phosphate, two removed, one phosphate bind to carbon 3 of sugar form a molecule of water

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15
Q

what is the Maxam-Gilbert method for sequencing a DNA molecule - functions

A
  1. based on chemical modification of DNA and subsequent cleavage at specific bases
  2. it is technically complex
  3. it uses hazardous chemicals
  4. it is difficult to scale it up
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16
Q

what HAPPENS in Maxam-Gilbert method for sequencing a DNA molecule

A

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

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17
Q

what method led to the methylation interference assay

A

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

18
Q

what is the methylation interference assay used for

A

to map DNA binding sites for DNA binding proteins

19
Q

why is the Sanger dideoxy method for sequencing a DNA molecule good

A

more efficient and uses fewer toxic chemicals and lower amounts of radioactivity

20
Q

what does the Sanger dideoxy method require

A

defined primer that binds to the 3’ end of the template strand to be sequenced

21
Q

what is the Sanger dideoxy method based on the use of

A

dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators

22
Q

what must the primer of the Sanger dideoxy method for sequencing a DNA molecule have

A

must be tagged at its 5’end with a radioactive 32P-phosphate (gel) or a fluorescent dye (automated sequencer reading)

23
Q

how is the DNA synthesised and how is it terminated in the Sanger dideoxy method for sequencing a DNA molecule

A

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

24
Q

how does the DNA synthesis occur in Sanger dideoxy method for sequencing a DNA molecule

A

incorporation of a 2’-, 3’-dideoxynucleotide, which leaves no 3’-OH group for further chain growth

25
Q

what does the Sanger dideoxy method for synthesising a DNA segment produce

A

series of fragments with various lengths will be generated which can then be used to read the nucleotide sequence

26
Q

what is do the four identical reaction mixtures require in the Sanger dideoxy method in the lab

A

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

27
Q

what is needed to support the 5’ to 3’ DNA synthesis in the Sanger dideoxy method in the lab

A

Klenow fragment of DNA polymerase I

28
Q

when does synthesis of new strand stop

A

Synthesis of the new strands continues until a dideoxyribonucleotide is incorporated

29
Q

what is used to detect the radioactive bands

A

autoradiography

sequence of the original template strand can be deduced from the bands on the autoradiograph

30
Q

what lowers the cost of DNA sequencing

A

High-throughput sequencing technologies

31
Q

examples of high-throughput sequencing technologies

A

illumina (solexa) sequencing, DNA nanoball sequencing

32
Q

what do we need to know about the genome

A
  1. how the genome is organised
  2. structure of genes
  3. function of genes
  4. how these genes are related
  5. how various parts of the genome are coordinated
  6. genes account for less than 25% of the DNA in the human genome
  7. unidentified secrets of life
33
Q

how do you sequence a genome

A 
break the genome into pieces either using 
1. clone-by-clone approach
2. whole-genome shotgun
sequence the pieces
reassemble them in proper order
34
Q

what do the non-protein coding sequences make up in prokaryotes and eukaryotes

A

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

35
Q

what does the genome map include

A

sort DNA sequences
regulatory sites
promoters
genes

36
Q

what is a genome map like

A

one-dimensional-it is linear; it looks like a straight line with landmarks noted at irregular intervals along it

37
Q

what are the landmarks in a genome map

A

combinations of letters and numbers that stand for genes or other features

38
Q

what is the use of a genome map

A

need map to understand the genome sequence
can help in the sequencing of a genome
help scientists study genes

39
Q

if us clone-by-clone method what is the map like

A

indispensable to determine where each clone belongs in the genome

40
Q

what are genome maps used for

A

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

Applied Genetics (20 decks)

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