OCR Biology A level 6.1.3 Manipulating genomes summary notes

6.1.3 Manipulating Genomes

Outline the steps involved in sequencing the genome of an organism;
Genome: all the genes possessed by an individual organism or the whole sequence of bases in all of the DNA in an
organism.
DNA sequencing: process of determining the order of nucleotide bases (A, T, G and C) in a molecule of DNA

Relies on PCR and dideoxynucleotides (ddNTPs)
 Same as nucleotides except they contains a hydrogen on the 3’
carbon instead of a –OH group.
 When integrated into a sequence they prevent the addition of
further nucleotides as it cannot form a phosphate bridge
 Contain a fluorescent marker – each one will be a different colour

A genome must be fragmented before sequencing – why?
i) Genotype too big; sequencing reaction can only operate on a length
of DNA of about 750 base pairs
ii) Accuracy better/fewer errors (with small fragments)
iii) Divide job over time/different labs

Similar to PCR BUT only ONE primer and deoxynucleotides instead of nucleotides.

Step 1:
 Into 4 eppendorfs (one for each variant of labelled dideoxynucleotides) add:
o DNA to be sequenced
o DNA polymerase
o Your primer
o Nucleotides – contains the four bases A, C, G & T
o One dideoxynucleotides (A/C/G/T)
 DNA replication will halt every time a modified base is used




Step 2:
 Add eppendorfs to thermocycler (PCR)
o Denaturation step
 Heated briefly to 95oC to denature DNA
 Hydrogen bonds break so the DNA double helix separates creating two single stranded DNA
molecules
o Annealing step
 Thermal cycler then cools down to 50 oC
 Primer attaches to one strand of DNA
 Tells DNA polymerase where to start adding nucleotides (will copy one strand but not the
reverse strand)

, o Extension step
 Thermal cycler changes the temperature to 72 oC
 DNA polymerase locates primer and attaches
 Nucleotides are added on to the grown chain by DNA polymerase
 Whenever a dideoxynucleotides is used instead of normal deoxynucleotide, DNA replication
stops – “Chain-terminating event”




If looking at one tube, e.g “G” tube – find a mixture of products ending with G dideoxynucleotide
 Each strand terminates at a different point depending on where the modified nucleotide was added
 Bands of all different lengths produced

Step 3:
 Electrophoresis
 Contents of each of the four tubes are run in separate lanes on a electrophoresis gel in order to separate the
different sized bands from one another
o Smaller fragments are produced when the ddNTP is added closer to the primer
o Chains are smaller and therefore migrate faster across the gel
o After the contents have been run across the gel, the gel is then exposed to either UV light or X-Ray
depending on the method used for labelling the DNA.

The complementary base sequence can be read from the gel - the smallest nucleotide is at the bottom of the gel.




Automated sequencing: more DNA can be sequenced in a shorter period of time.
 Reactions are performed in a single tube containing all four ddNTPs each labelled with a different colour dye
 DNA separated on a gel by electrophoresis
o All run on the same lane
o Shortest lengths move fastest and the longer ones move slowly
 Since the four dyes fluoresce at different wavelengths, a laser then reads the gel to determine the identity of
each band according to the wavelengths at which it fluoresces.
 Results are shown on a chromatogram (diagram of colored peaks that correspond to the nucleotide in that
location in the sequence)