Genetics- Manipulating Genomes Flashcards
What is a genome?
All of the genetic material an organism contains
What is satellite DNA?
Short sequences of DNA that are repeated many times
What is minisatellite and microsatellite DNA?
- Smaller sequences of DNA repeated less times than the previous
- Mini: 20-50 base pairs repeated 50 to several hundred times
- Micro: 2-4 base pairs repeated 5-15 times
How do satellite patterns work?
- The more closely related you are to someone, the more likely you are to have similar patterns
- Satellites always appear in same position of chromosomes, however number of repeats varies
- Different lengths of repeats inherited from both parents
What is DNA Profiling?
Producing an image of the patterns in the DNA of an individual
What is DNA profile used for?
- Identification of individuals in forensics
- Paternal or familial testing
How to produce a DNA profile?
1) DNA must be extracted from a tissue sample. Samples can be small due to the use of PCR
2) Strands of DNA cut into small fragments using restriction endonucleases, which make two cuts through each strand of double helix
3) Cut fragments of DNA separated using electrophoresis, using charged particles that move through a gel medium under the influence of an electric current
4) Gel is then immersed into alkali to separate double strands into single strands
5) Radioactive or fluorescent DNA probes added in excess to DNA fragments. They bind to complementary strands (hybridisation). DNA probes identify the microsatellite regions that are more varied
6) X Ray images taken or sample placed under UV light. Fragments give a pattern of bars, which is the DNA profile that can be used to identify a suspect or show familial relationships
What is PCR?
- Polymerase Chain Reaction
- Used when only very tiny amounts of DNA might be available
- PCR is the process by which DNA is replicated, creating a sample large enough to be analysed
How does PCR work?
1) Temperature in machine at 90C, which denatures DNA, breaking the hydrogen bonds holding the two strands together so they separate
2) Temperature is decreased and the primers anneal (bind) to the ends of the DNA strands to allow replication to occur
3) Temperature increased again to 70C, which is the optimum temperature for DNA polymerase to work. DNA polymerase adds bases to the primer, building up complementary strands of DNA, producing double-stranded DNA identical to original sample.
4) Process continues for about 30 repeats
What enzyme is used for PCR and why?
Taq polymerase because it originates from thermophilic bacterial and so won’t denature at the high temperatures required for PCR
What is DNA sequencing?
Process of determining the precise order of nucleotides within a DNA molecule
How is DNA sequenced?
1) DNA for sequencing mixed with primer, DNA polymerase, an excess of normal nucleotides and terminator bases
2) Mixture placed in thermal cycler which rapidly changes temperature and the double stranded DNA gets separated in single strands and the primers anneal to the DNA
3) DNA polymerase builds up new strands by adding nucleotides by complementary base pairing
4) Each time terminator base is incorporated, instead of a normal nucleotide, the synthesis of DNA is terminated. As terminator bases are added at random. it results in many DNA fragments of different lengths. After many cycles, all the possible DNA chains will be produced with the reaction stopped at every base
5) DNA fragments separated by length by capillary sequencing, which works like gel electrophoresis in minute capillary tubes
6) Lasers detect the colours and thus the order of the sequences. This can be used to build the sequence of the original DNA as it is fed into a computer which reassembles the genome by comparing all fragments and looking for areas of overlap
How has DNA sequencing changed over time?
-DNA sequencing technologies have become faster and more automated
-Sequencing reactions take place on a plastic slide, known as a flow cell
-Millions of fragments of DNA attached to slide and replicated using PCR to form clusters of identical DNA fragments
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