Molecular Techniques to study DNA and RNA Flashcards
What is the functions of genetic material?
-It must replicate the growth and development of the organism and allow the organism to adapt to changes in the environment
What is proof that DNA is genetic material?
-griffith’s experiment
-4 methods
1.Type IIS (virulent) bacteria are injected into a mouse
-proves that type 3s causes death bacteria can be recovered
-b) type IIR non virulent bacteria are injected into a mouse
-no bacteria can be recovered because mouse can destroy pathogen
-c) heat killed type IIS bacteria are injected into a mouse
-this is the same as a only difference is that the virus was boiled
-d) a mixture of type IIR bacteria and heat-killed type IIS bacteria are injected into a mouse
-a substance in the heat-killed virulent bacteria genetically transformed the type IIR bacteria into live virulent type IIS bacteria
What is the Avery-MacLeod- McCarty experiment?
-narrowed it down to DNA
-methods: type IIIS (virulent) bacteria
Use heat to kill virulent bacteria and filter
-2 Type IIIS bacterial filtrate treat samples with enzymes that destroy proteins RNA or DNA
-RNase destroys RNA protease (destroys proteins) transformation is still happening
-DNase-destord DNA
-add treated samples to cultures of type IIR bacteria
Cultures treated with protease or RNase contain transformed type IIIS bacteria but the culture treated with DNase does not
-concluded DNA virus because was not present after DNase
What substance, RNA or protein, carries the genetic material in tobacco mosaic virus (TMV)?
-type A TMV and TB TMV
-degrade both types of TMA to yield RNA and coat proteins
-mix RNA of one typw with protein of the other
-are then swapped cores and shells which created hybrid viruses infect tobacco with the hybrids
-results: the Type of RNA in the hybrid parent TMV determines the RNA and protein of the progeny viruses thus RNA is the genetic material of TMV
What are nucleic acids composed of?
-nucleotides joined by phosphodiester links C-3 o f one sugar to C-5 of another sugar
-the same bond is found in RNA
-nitrogen-containing bases
-purines: Adenine, Guanine,
-pyrimidines: Thymine Cytosine, Uracil)
-purines and pyrimidines
-sugar (pentose) deoxyribose (H)
-ribose OH
-phosphate (phospho-diester bond)
How was the structure of DNA deciphered?
-Erwin Chargaff (1949-1953) determined the precise base composition of different types of DNA
-%A = %T %G = %C
A + G = T + C
purines = pyrimidines
-William Astbury (1947): X-ray diffraction Analysis showed that DNA is a polymer of stacked bases
-Rosalind Franklin & Maurice Wilkins (1950 - 1953) :DNA is a HELIX
What is the The Watson - Crick Model (1953)?
-one of the most celebrated discoveries in science
Background for the model:
- % A = %T
- % G = % C
-Helical structure
-Bases stuck like coins over each other
-Assumptions:
-DNA is a double helix
-The two strands were anti-parallel
-The sugars form a phosphate backbone
-The bases are held together by H bonds
2 H-bonds between T & A
3 H-bonds between C & G
What are the key points: DNA Structure?
- Complementary Base Pairs (A with T, G with C)
- Antiparallel Strands
- The two strands of the helix have opposite chemical
polarity (5’ and 3’ ends)
What are the techniques for DNA and RNA Analysis?
-Molecular hybridization:
-gel electrophoresis
Restriction enzymes and cloning
-polymerase chain reaction
-next generation sequencing
What is molecular hybridization?
-fluorophore (donor) quencher (acceptor) where hybridization occurs
What is gel electrophoresis?
-reaction between cathode and anode where liquid will get solid
-restriction enzyme cleaves DNA at different spots
-DA samples are loaded into wells
-power is turned on and DNA fragments migrate through gel
-phosphate group makes DNA highly negatively charged
-largest flows through gel slower and smaller flows through gel faster
-fragments are separated by charge
What are restriction enzymes and cloning?
Restriction enzymes (RE) are a group of endonucleases produced in bacteria as a means of destroying foreign DNA
-cleave viral DNA in immune system
They function by cleaving DNA at specific double stranded sequences known as restriction sites.
Restriction sites are typically 4 - 8 bp in length and are often palindromic.
These sites occur randomly and at many distinct locations throughout the genome.
As such RE can be used as a tool to target and cleave DNA at the particular locations where a restriction site occurs.
-EcoRI same formed backword and backward the sticky ends cuts between G and C
-have hydrogen bonding potential
-SmaI has blunt ends with no sticky ends no hydrogen bonding and will not stick back together
What are the three components of a cloning plasmid?
Naturally occurring in bacteria and yeast, many plasmids have been edited and engineered to be
optimized for use in cloning. A cloning plasmid requires three components:
1) a Multiple Cloning Site (MCS), which is a region with many restriction sites into which
-contains restrictive enzyme sites
-gives flexibility to use whichever enzyme they want
exogenous or external DNA is inserted
-2) an origin of replication (ORI) site which enables
plasmid replication in host cells;
3) a selectable marker (eg. antibiotic resistance gene ampr)
which enables researchers to select cells that contain the plasmid
What are the four steps involved with fragment of interest?
- Digest: plasmid and foreign DNA are cleaved with restriction Enzymes forming complementary sticky ends which cuts open and is placed
-must have ORI to be competitive with other restriction enzymes and plasmids is deposited
-2 Ligation: DNA fragment and plasmid hybridize at sticky ends
DNA ligase forms phosphodiester bonds to seal nicks in each strand
-plasmid can close on itself because of the sticky ends before accepting the sticky en s
-the p HO makes the structure stable - Transformation: teh ligated plasmid simixed with bacterial cells under conditions to optimize transformations
-4: Selection only cells containing the plasmid will grow on ampicillin plates forming colonies which all have plasmid
What is Polymerase Chain Reaction?
-used in crime
-Originally developed by Kary Mullis (1944 - 2019) the process of PCR revolutionized the field of genetics and earned
Mullis the Nobel Prize in Chemistry (1993, shared with Micheal Smith).
-This technique builds on the natural process of
DNA replication (covered in the 2 nd half of MBG*2040) as well as concepts of DNA hybridization to target and
replicate specific DNA sequences producing large quantities of the target DNA
-Because this process is quick, inexpensive, highly versatile and requires very little starting material it is an extremely
powerful tool in molecular biology.
