Summary Biology A2 level 'application of reproduction and genetics' notes (WJEC)
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Course
Application of reproduction and genetics
Institution
WJEC
Biology A level 4.5 'application of reproduction and genetics'. These notes were formulated using class notes, the WJEC study guide and past papers. These notes helped me to achieve an A* at A level biology!
Human genome project
● Aims;
○ Using the Sanger sequencing, identify all genes in the human genome and
identify their loci
■ Loci = position on the chromosome
○ Determine sequence of the 3.6 billion bases in the human genome and store in
databases
○ Consider the ethical, social and legal issues that arise from storing such
information
● Findings;
○ Number of genes present in the human genome is around 20,500
○ There are large numbers of repeating sequences, called STRs (short tandem
repeats)
Sanger sequencing method
● Sequences small fragments of DNA (800 bases in length) created by restriction enzymes
○ Restriction enzymes - bacterial enzymes that cut DNA at specific base
sequences
● DNA polymerase used to synthesise complementary strands, using the polymerase
chain reaction
○ Polymerase chain reaction - rapidly produces a large number of copies of a
specific fragment of DNA
○ Four reactions were carried out (for adenine, thymine, cytosine, guanine)
■ Contained complementary nucleotides marked with a radioactive marker
■ Some nucleotides had been altered to stop nucleotides ⇾ prevent further
synthesis
● E.g. 5’ ACGTAGCCCGGTAG 3’
○ Random whether a normal thymine nucleotide or a stop nucleotide is
incorporated
○ Some DNA strands will have incorporated a stop nucleotide, others won't
○ Some strands will be 4 bases long, others 12 bases long
○ Results for all reactions are run side by side on an agarose gel using
electrophoresis
■ Electrophoresis - separates molecules according to size
■ Exposed to x-ray film to detect the radioactive signal
■ Sequence can be determined by reading the banding pattern
● Slow method, takes days to sequence a few thousand bases
○ Next Generation Sequencing (NGS) entire genomes can be sequenced in hours
, Gel electrophoresis
● Method of separating DNA fragments according to size
● Gel is made from agarose (polysaccharide) & contains pores in its matrix
● Method
○ DNA is extracted from the sample and cut into small fragments using restriction
endonucleases
■ Restriction endonuclease - bacterial enzyme which cuts DNA at specific
nucleotide sequences
○ DNA samples are loaded into wells at the end of a trough containing gel
○ Voltage is applied across the gel
○ DNA is attracted to the positive electrode due to the negative charge on the
phosphate groups
○ Smaller fragments migrate more easily through the pores ⇾ travel further than
large fragments in the same time, so DNA becomes separated according to size
■ Size of fragments can be estimated if a sample of known DNA sized
fragments (a DNA ladder) is run at the same time as the samples
● DNA probes can be used to find DNA sequences of interest within DNA fragments
○ Probe = short piece of single stranded DNA that's labelled with a fluorescent or a
radioactive tracer (32P)
○ Probes are complementary to the part of the sequence of interest
○ When the probe is washed over the gel it binds to exposed complementary
nucleotides (complementary base pairing) by a process called DNA hybridisation
○ DNA fragment of interest is identified by its fluorescent / radioactive signal
■ To detect a radioactive signal;
● DNA is transferred to a nylon membrane
● Exposed to X-ray film ⇾ produces an autoradiograph
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