Polymerase Chain Reaction and Alu Elements Flashcards
What are the steps in a recombinant DNA study?
- Amplify gene of interest.
- Insert gene into vector.
- Insert recombinant DNA into living cells to propagate it.
- Restriction endonucleases are used in the process.
What is PCR?
Polymerase chain reaction is a technology that allows specific DNA sequences to be amplified.
What are the three major steps of PCR?
- Denaturation
- Annealing
- Extension
Denaturation
target DNA is denatured and the DNA strands are separated.
1 min 94 degC
Annealing
2 short oligonucleotide primers are annealed to the denatured DNA. Primers are complementary to the two 3’ ends of the DNA to be amplified.
45 seconds 54 degC
Extension
DNA Polymerase adds nucleotides extending the primers. Two duplex DNA copies are generated from the template.
2 mins 72 degC
How is the number of cycles related to the number of amplification?
DNA is amplified (2)^number of cycles
What is Taq I Polymerase and what is special about it?
a thermostable DNA polymerase, can be heated to high temperatures.
What is a thermocycler?
machine where PCR reaction is conducted.
What are some applications of PCR?
production of probes, gene cloning and manipulation, gene mutagenesis, DNA sequencing, amplification of ancient DNA, diagnostic in medicine, and forensic DNA typing.
What are non-coding sequences?
self replicated, selfish, no function expect their own reproduction. Ex: Alu elements
Where are Alu elements found?
Found in exons, but mostly exist in introns and other non-coding regions.
How are Alu elements replicated?
replication is through an RNA intermediate, copied into dsDNA called retrotransposon that inserts randomly elsewhere in the genome.
Polymorphisms
DNA sequence which varies between individuals.
Dimorphic
insertions that might be present or absent in each homologous chromosomes.
Is the Alu sequence dimorphic?
yes
Where is the Alu sequence found in the chromosome?
found on a section of chromosome 16 PV92 locus.
How many base pairs is chromosome 16 PV92 locus?
this section of DNA is 700 bp, insertion of Alu sequence (300 bp) increases the length of the region to 1000 bp.
What does one band at 1000 bp indicate on agarose gel?
Individual is homozygous for Alu insertion
What does one band at 1000 bp and second band at 700 bp indicate on agarose gel?
Individual is heterozygous for Alu insertion
What does one band at 700 bp indicate on agarose gel?
Individual is homozygous for no Alu insertion
What is gel electrophoresis of DNA?
used to monitor DNA cleavage at alkaline pH.
What charge is DNA and RNA?
negatively charged.
What size of DNA fragments is PAGE used for?
small DNA fragments.
What size of DNA fragments is agarose gel used for?
check larger DNA molecules, separation depends on length of DNA molecule.
