Lab 4 Flashcards
How is human DNA unique
although 99.9% of human DNA sequence is same in every person, there are enough differences in the genomic DNA that makes each person so unique
these differences include: differences in restriction sites. Note. no two individuals will have the same DNA profile unless they are monozygotic twins
restriction enzymes
proteins that cut DNA at specific sites called restriction sites
they possess a 3 dimensional structure necessary for them to function properly
restriction sites
restriction sites have specific sequence combination that can be recongnized by specific restriction enzymes
the restriction enzyme cuts both strands of DNA molecule
most restriction sites have palindrome sequences.`
what do restriction sites recongnize?
they recongnize the palindromes between the COMPLEMENTARY DNA STRANDS, NOT on the single strand
Restriction sites have what type of end options
sticky ends and blunt ends
number of fragments after digestion
restriction enzyme recognizes DNA sequences from 4-8 base pair in length
the greater the number of bases required, the fewer fragments produced
4-cutter enzyme recognizes restriction sites with 4bp sequence combination, such as GATC
6-cutter enzyme recognizes 6bp site, GAATTC
how do you know the number of fragments after digestion
X^n
X= the number of bases (AGTC =4 mostly), n=bases recognized by enzymes
what are some factors affecting the protein conformation (which alters activity?)
ionic concentration
optimal pH
temperature
what are ways to ensure optimal protien function
ddH2O and clean reagents, buffer, 37C
what restriction enzymes were used in lab?
EcoRI
Hindlll
PstL
agarose gel
a polysaccharide polymer, extracted from seaweed
in a gel it forms matrix that allows for differential migration of differently sized DNA fragments
basically it forms a complicated molecular network that DNA fragments can flow through at different speeds depending on the size of the DNA
with more agarose the matrix become more complex and the DNA fragments migrate slower
what factors affect migration rate?
- size of DNA=the larger the DNA fragment, the slower it migration will be
- agarose concentration=the greater the concentration, the denser the molecular matrix, the slower the DNA will migrate
- voltage= the greater the voltage used, the faster DNA will migrate
- buffer strength=the greater the concentration of electrolytes, the faster the DNA will migrate
how does concentration of agarose concentration affect?
- higher the concentration of agarose, better is the resolving power of the gel (if two fragments that have very small difference in terms of their size)
- PCR amplifies small fragments of DNA ( amplifies maximum of 1500 bp effectively). Hence a denser matrix (higher agarose concentration) is used to provide a better resolution to separate the smaller fragments of DNA amplified by PCR.
Loading dye
samples are mixed with a loading dye before”loading” them into wells of gel
loading dye has two components: that have important functions
- dye=which will help visualize migration of DNA (which is invisible)
- glycerol= which will weigh the sample down and make it easier to put into the wells
DNA marker/ladder
contains a standarized set of different sized fragments
provides a reference for determining the relative size of a DNA fragment
