The PCR and DNA profiling Flashcards
1
Q
What can the PCR samples be used for in DNA sequencing?
A
- Paternity testing and forensic science (to identify criminals) to build a DNA profile
- to predict the amino acid sequence of proteins
- Can be used in DNA sequencing, where its possible to identify faulty genes and its links to genetically determined diseases/conditions.
2
Q
How can the PCR amplify DNA samples?
A
- DNA sample, DNA polymerase, primers and nucleotides are added into a PCR machine.
- Mixture is heated to 95C causing DNA strands to separate (hydrogen bonds broken)
- Cooled to 55C for primers to bind to their complementary base pair on the DNA strands (annealing)
- Heated to 72C so DNA polyermase can build up the complementary strands
3
Q
How is a DNA profile produced.
A
- Once DNA has been extracted and amplified by the PCR, RESTRICTION ENDONUCLEASES cut the samples at RECOGNITION SITES, producing different sizes fragments.
- Gel electrophoresis separates the fragments with a dye that flouresces under UV light so the bands are visible. An electric current passes through (DNA moves to anode because of negative charge on phosphate). Small fragments move further along gel because they have less resistance to the gels pores.
The banding pattern is known as the DNA profile.
4
Q
How is DNA sequenced ?
A
- DNA molecules are cut into fragments and amplified by the PCR then separated into separate strands.
- Strands are further replicated using a mixture of of bases with TERMINATOR BASES (have fluoresced tags that stop replication at a specific base)
5
Q
What is the Southern Blotting method?
A
Alkaline solution is added to the gel after electrophoresis and a nylon filter is placed over the gel which picks up fragments and blots on the paper.
The solution denatures the DNA fragments so strands separate, exposing base sequences.
Gene probes are added to the filter and bind to complementary strands - when the filter is placed under UV lights the DNA regions are visible.
