Applications of Replication Flashcards
Polymerase Chain Reaction
technique that allows millions of copies of DNA to be amplified from small starting samples
How does the PCR work?
- sample of DNA is placed into a tube with a buffer solution containing essential ions and salts and a pair of DNA primers
- primers will bind in a complementary manner to specific regions of template DNA and serve as a starting point for DNA copying
- Free deoxyribonucleotides are added to the tube
- placed in thermocycler which heats and cools the solution
- Taq polymerase is added to cataylze the polymerization of each daughter strand
Denaturation
-reaction mixture is heated to denature the strands of double stranded DNA
Annealing
- thermocycler will cool the solution to allow the primers to anneal to their complementary sequences on the DNA template strands
- primers will bind on opposite strands at each end of the target sequence
Extension
-heat stable DNA polymerase extends and polymerizes the daughter strands using the four dNTPs, starting from the primers and then extending in the 5-3’ direction
PCR results in ____________
two double stranded helices containing the desired target sequence portion of the original template DNA
Gel Electrophoresis
general technique used to separate DNA fragments from other sources
How Gel Electrophoresis Works
- goes through an agarose gel in an electric field
- molecules are loaded into wells of a porous gel
- move because of electric field
- DNA is a negative molecule and will be attracted to positive (anode side)
- smaller the fragment the further they travel
- ladder with known size is loaded
Shot gun Sequencing
- meyers and Webber came up with it
- able to break the entire genome into different size pieces and split it into three phases
Sequence DNA
- all components for DNA replication are needed for sequencing (DNA polymerase, dRTPs, primers and template DNA
- insertion of ddNTPs is random and theres less added than dNTPs
- allows for many different sized fragments
- each of the four ddNTPs can be labelled with different fluorescent dyes and tracked in gel electrophoresis
Dideoxynucleotides
would prevent further elongation because it is missing the OH group making it unable to bond to the next nucleotide
Assemble Sequences
-identifying the regions of overlap between the generated fragments and assembling the long continuous sequence of nucleotides in the DNA molecule that makes up each chromosome
Significance of Assembling Sequences
- allow for the information from multiple DNA sequences to be assembled by examining the regions of overlap between random DNA fragments
- assembly of final DNA is similar to putting together puzzle pieces
- sequences of entire genomes can be determined based on the similarities based on the overlaps
Contigs
overlapping DNA segments that are assembled into a consensus region of DNA
-assembly of these fragments is done by complex algorithms
Annotate the Sequence
-annotate the sequences to identify the regions of DNA that encode genes, regulatory regions and even non-coding regions of DNA
