manipulating genomes Flashcards

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1
Q

what is DNA sequencing

A

working out the sequence/order of nucleotide bases

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2
Q

what is meant by the term genome

A

the entire genetic material of an organism

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3
Q

why is knowledge of DNA sequences useful

A

understand evolutionary relationships, comparing genomes between individuals and species and to be able to predict amino acid sequences

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4
Q

what is the chain termination method - manual sanger sequencing

A
  • uses modified nucleotides called dideoxynucleotides (terminator nucleotides)
  • these pair with nucleotides that have a complimentary base
  • when DNA polymerase encounters a dideoxynucleotide on the developing strand it stops replicating
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5
Q

what do dideoxynucleotides do

A

stop replication as it cannot form a phosphodiester bond between it and a nucleotide

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6
Q

what is different between the structure of deoxynucleotides compared to terminator nucleotides

A
  • have less hydroxyl groups
  • have no condensation reaction
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7
Q

what is the process of manual singer sequencing

A

1- many copies of a single stranded piece of DNA are used as templates to sequence
2- a primer is added so that DNA polymerase can bind and begin copying the DNA strands
3- the DNA and primers are added into 4 identical test tubes with free normal nucleotides, for DNA polymerase to add to the growing chain a small amount of terminator nucleotides are also added randomly
4- in each tube many copies of the DNA molecule are made but all different lengths
5- the 4 tubes are then loaded into agar jelly to undergo electrophoresis
6- by reading the pattern scientists can work out the order of bases in the DNA sequence

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8
Q

what is the process of automated sanger sequencing

A

1- each type of terminator nucleotide is labelled using a specific fluorescent dye
2- the single stranded DNA chains are separated according to mass using capillary electrophoresis (works same as electrophoresis but is just in capillary minute tubes)
3- a laser beam is used to illuminate all of the terminator nucleotides and a detector then reads the colour and position of each fluorescence
4-the detector feeds the information into a computer where it is stored or printed out for analysis

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9
Q

what are the uses and strengths of sanger sequencing

A
  • gives high quality sequence for relatively long stretches of DNA (up to 900 base pairs)
  • it is typically used to sequence individual pieces of DNA such as bacterial plasmids or DNA copied in PCR
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10
Q

what are limitations of sanger sequencing

A
  • expensive and inefficient for larger scale projects e.g. sequencing of an entire genome
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11
Q

what is next-generation sequencing

A
  • any method of DNA sequencing that has replaced the sanger method
  • a set of massively parallel sequencing technologies that allow for the simultaneous sequencing of many DNA fragments in a high throughput manner.
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12
Q

what is next generation sequencing also known as

A

high-throughput sequencing

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13
Q

what is different about NGS and the sanger method

A
  • NGS can generate millions or billions of short DNA sequences in a single sequencing run where as the sanger method which sequences one fragment at a time
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14
Q

NGS in comparison to sanger method is …

A
  • highly parallel and fast as many sequencing reactions take place at the same time
  • micro scale as reactions are tiny and many can be done at once on a chip
  • low cost as it is much cheaper
  • shorter length as it reads typically range from 50-700 nucleotides in length
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15
Q

what is the system of pyrosequencing

A

1- a piece of DNA is cut up into fragments (400-600bp) using restriction enzymes
2- they are made single stranded and are the template DNAs, they are immobilised on a bead
3- a primer is added and DNA incubated with DNA polymerase and other enzymes
4- then one of the 4 specially activated nucleotides are added at any one time
5- if the next nucleotide is attached, there is a reaction and light is emitted which can be detected by a camera, if 2 of the same nucleotide are next to each other double the light is emitted
6- any unused nucleotides are degraded before the next base is added

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16
Q

what is nanopore sequencing

A
  • currently being developed and invloves single strands of DNA are pulled through a pore (as each each base is pulled through the pore its conductivity is measured). Any length of DNA can be sequenced
17
Q

what are uses of DNA sequencing

A
  • human genome project
  • comparing the genome of individuals
  • comparisons between species
  • evolutionary relationships
  • epidemiology
18
Q

what are the 3 stages of PCR

A
  • denaturation
  • annealing
  • extension
19
Q

what does denaturation involve

A
  • heating the section of DNA to 94-96°c through the use of a thermal cycler
  • this breaks the hydrogen bonds between the 2 strands
  • leads to single stranded DNA
20
Q

what does annealing involve

A
  • cool both strands to 54-60°c
  • and primers added
  • the cooling allows the primers to bind to the strands
21
Q

what are primers

A
  • short strands of DNA that are complimentary to the bases at the start of the target fragment
  • tell DNA polymerase where to attach
22
Q

why is taq polymerase used

A
  • doesn’t denature at high temperatures
  • many cycles of PCR can be carried out using the same enzyme
22
Q

what does extension involve

A

-heated to 72 and taq polymerase attaches
-72° is the optimum temperature
- new strands are formed and the target DNA is doubled

23
Q

what is the process of electrophoresis

A
  • DNA fragments are loaded into wells
  • an electric current is passed through the electrophoresis tank which pulls the DNA fragments through the agar jelly towards the anode as DNA is negatively charged
  • this separates the DNA fragments into size
  • DNA fragments of the same length form a band that can be seen by the eye if it has been stained with a DNA-binding dye
24
Q

what are the applications of PCR

A

-forensics
-genetic testing
-diagnostics
-genetic disorders