Molecular genetics p2 Flashcards
Recombinant DNA
DNA that’s been prepared in the laboratory by combining fragments of DNA from more than one source
Restriction endonucleases/enzymes
They cleave double-stranded DNA from within. They do so by recognizing a short sequence of nucleotides, called the target sequence within the DNA.
Recognition site (aka restriction site)
Enzymes cut the strand at a particular point in the sequence called the restriction site
Restriction enzymes video
- Plasmids are small loops of DNA in bacteria
- Restriction enzymes recognize specific sites in DNA to cut
- Sticky ends will H+ bond to complimentery nucleotides, a seperate peice of comp DNA can bond if it also has sticky ends. Ligase joins the ends. Result is recombinant DNA.
- Sequence specificity –> targets specific sequences
- Recombinant DNA can be used to breed a specific gene in bacteria, then take it out
- Plasmid and gene fragment joins in phosphodiester linkage
PCR
- used to copy DNA fragments or genes
- a primer attaches on either side of a target, one primer binds to each strand through complimentry base pairing (they’re custom per target)
How does PCR machine work?
- 95 celcius disrupts complimentry base pairing, DNA strands separate
- 50 celcius, primers bond to strands instead of helix reforming
- 72 celcius activates DNA polymerase, attached to end of primers and extends them –> adds complimentry nucleotides along single stranded DNA - goes until it falls off, reaches end of strand or next temp change
- 30 cycles, over a billion copies of target sequence, 60 of longer sequence
Uses of PCR
- detects harmful bacteria
- indefies viruses
- tests for inherited disease
-paternity testing - indentify a species
- detect species in water
- connect people to crime scenes
- learn about human history
-learn about extinct species
Gel electrophoresis
- seperates DNA of different lengths, other molecules like proteins will seperate
- adding an electrical current over the gel, DNA goes negative to positive
- shorter strands more quicker so they will be farther towards the positive end
- DNA is dyed so it can be seen
- Gel is made of Agrose and buffer liquid
- DNA ladders are measures for the number of base pairs in DNA sample (most –> least)
- Ethidium bromide us used to stain gel after, it absorbs UV, but its also a highly toxic mutagen
DNA fingerprinting (DNA profiling)
- Identification of people based on their DNA (because each of us has a unique DNA sequence)
- Traditionally used a technique called RFLP = Restriction Fragment Length Polymorphism which used restriction enzymes (to cut DNA into different sized fragments) and gel electrophoresis
- Now called STR profiling (short tandem repeats), selective PCR primers eliminated the need for restriction enzymes
How does STR profiling work?
- part of the variation between people occurs in areas of DNA that do not contain genes coding for protein
- They are highly repetitive DNA because they contain groups of nucleotides that are repeated over and over
- These are called STRs (short tandem repeats)
- The repeats vary in length depending on how many copies of the STR are present. The more there are, the longer the fragment
*humans are 99.9% the same - primers are designed to amplify specific regions. These regions vary in length between people. This difference in length is called a polymorphism and is unique to the person
- Due to the length difference of the pcr products, gel electrophoresis can be used to analyze and compare samples
Basic steps STR profiling
- sample collected
-DNA extraction - PCR
- Gel electrophoresis
- Comparison of samples
How good is the DNA fingerprint
Its possible people will share the same banding pattern for a small, specific area if:
- indentical twins
- close genetic relatives (parent/child/sibling)
Application STR profiling
- forensics
- ID of victims
- ID of genetic parents
What is DNA sequencing
- the process involved in determining the exact sequence of nucleotides in a piece of DNA
- the human genome project used computer technology to read the human DNA sequence AND this technology is based firmly on the gene sequencing laboratory techniques developed over the past two decades
What is a dideoxynucleotide triphosphate?
- a regular deoxynucleotide is missing an oxygen at only the 2 prime carbon
- A dideoxynucleotide is missing an oxygen at the 2 and 3 prime carbons (3 prime is needed to form a phosphodiester bond with next nucleotide)
- DNA polymerase 3 therefore, can’t add another nucleotide
- if a dideoxynucleotide got incorporated, DNA replication would stop
Frederick Sanger
- Developed the dideoxy termination sequencing method in 1975
- Based on the process of DNA replication that uses dideoxynucleotide triphosphates
- most popular sequenceing method, most automated
