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Summary 6.1.3 Manipulating Genomes Revision Notes (OCR A) $3.88
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Summary 6.1.3 Manipulating Genomes Revision Notes (OCR A)
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  • A Level Biology A for OCR Student Book

Comprehensive study guide for Biology A Level, made by an Oxford Biochemistry student with all 9s at GCSE and 3 A*s at A Level! Information arranged by spec point. Concise notes written using past papers, multiple textbooks, class notes and more.

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  • 6.1.3 manipulating genomes
  • April 8, 2021
  • 10
  • 2020/2021
  • Summary

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  • biology
  • ocr

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6.1.3 MANIPULATING GENOMES
a. the principles of DNA sequencing and the development of new DNA
sequencing techniques
Rapid advancements in gene sequencing:
 Sanger sequencing was the first method used to sequence long lengths of
DNA.
o It was efficient and safe, however it was also extremely time-
consuming.
 Nowadays we have new gene sequencing techniques that are much faster
and cheaper, and enable the sequencing of whole genomes.
o These are called high-throughput or next generation sequencing.
o One example is pyrosequencing.

Sanger sequencing:
 AKA chain termination sequencing.
 Take a single strand of DNA from the sample of interest.
 Cut the DNA into fragments of varying lengths (to max 500 base pairs) using
restriction enzymes.
o A genome must be fragmented before sequencing. It is too large, so it
will be difficult to distinguish between DNA molecules during gel
electrophoresis.
o Fragmenting the DNA makes sequencing more accurate.
 Add modified versions of each base and use PCR to amplify the DNA.
o Also add DNA polymerase, forward and reverse primers, free
nucleotides.
o These DNA bases are terminated nucleotides – once it is incorporated
into the complementary strand of DNA, no more bases can be added.
o The modified bases are also labelled with a radioactive isotope.
 The DNA fragments are then passed through a gel by electrophoresis.
 The nucleotide base at the end of each fragment is read according to its
radioactive tag.
 The sequence of bases can be pieced together using the different fragments.
The first DNA sequencing machine:
 Used fluorescent dyes instead of radioactivity to label the terminal bases.
 These dyes glowed when scanned with a laser beam.
 The light signature was identified by computer and used to sequence DNA
lengths.
Pyrosequencing:
 Uses sequencing by synthesis, not by chain termination.
 Involves synthesising a single strand of cDNA, one base at a time, whilst
detecting by light emission, which base was added at each step.
 The long length of DNA to be sequenced is mechanically cut into fragments
using a nebuliser.

,  These fragments are degraded into single-stranded DNA (ssDNA).
 The ssDNA fragments are immobilised on microbeads.
 The sequencing primer is added and the DNA is incubated with DNA
polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate
(APS) and luciferin.
o Enzymes: DNA polymerase, ATP sulfurylase, luciferase, apyrase.
o Substrates: adenosine 5’ phosphosulfate (APS), luciferin.
 One of the four possible activated nucleotides (ATP, TTP, CTP, GTP) is added.
 If the activated nucleotide is complementary to the ssDNA, it will be
incorporated into the cDNA.
o Two extra phosphate groups are released as pyrophosphate (PP i).
o In the presence of APS, ATP sulfurylase converts PP i to ATP.
o In the presence of ATP, luciferase converts luciferin to oxyluciferin.
 This generates visible light, which is detected by a camera.
 The amount of light generated is proportional to the amount of ATP available.
o Thus, it indicates how many of the same type of activated nucleotide
were incorporated adjacently into the cDNA strand.
 Each activated nucleotide generates a different colour of light.
o This enables the computer to read the base sequence of the DNA
fragments.
 Unincorporated activated nucleotides are degraded by apyrase.
 The process is repeated using the other activated nucleotide bases until the
entire fragment is sequenced.
More info about restriction endonuclease enzymes:
 Cut DNA at specific recognition sites (4 to 6 base pairs long).
 Made by bacteria and archaea to protect against phage virus attack.
o Cut up the foreign viral DNA to prevent the virus from making copies.
o The prokaryotic DNA is protected from the action of these
endonucleases by being methylated at the recognition sites.
 The recognition sites are always palindromic (e.g AGGCCT).
o Both strands have the same base sequence when read in the 5’ to 3’
direction.
 Some make a staggered cut, leaving sticky ends.
 Others make a straight cut, leaving blunt ends.
 Some need Mg2+ as cofactors.


b. i. how gene sequencing has allowed for genome-wide comparisons
between individuals and between species
Bioinformatics and computational biology:
 Involves developing software and computing tools to organise and analyse
the raw data.
 Compare the gene sequences between individuals.
o Identify genotype-phenotype relationships.
o Detect SNPs which make people more susceptible to diseases.
o Epigenetics (mapping DNA methylation) can be used to determine the
development of certain diseases, e.g. some cancers.
o Epidemiology – analysing the gene sequence of pathogens.

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