Techniques in Molecular Biology

Genetic Engineering - Efforts to manipulate DNA sequences in organisms

Goals of Genetic Engineering

• Improve our understanding of how genes work
• advance biotechnology - the manipulations of organisms to create products or cure disease

An Example - the Use of Recombinant DNA Technology to Produce a Save Supply of Growth Hormone

• Pituitary dwarfism - a disease caused by the lack of growth hormone (created by the gene GH1)
• Early research showed that the condition could be treated w/ injections of human growth hormone
  • Growth hormone could only be obtained from human pituitary glands
  • These were obtained from cadavers
  • Later studies showed that the cadaver supplied growth hormones were often contaminated, so other methods needed to be developed to artificially produce human growth hormone
• Use of Recombinant DNA Technology to Produce a Safe Supply of Growth Hormone
  • Isolate mRNAs from cells in pituitary glands
  • use reverse transcriptase to synthesize cDNA from each mRNA
  • Attach restriction endonuclease recognition site to ends of each cDNA
  • Cut cDNAs and plasmids w/ restriction endonucleases - remaining sticky ends join by complementary base pairing
  • Ligate cDNAs and plasmids w/ ligase
    • plasmids also contain antibiotic resistance gene
  • Introduce recombinant plasmids into E. coli cells to create a cDNA library
    • grow colonies on plate w/ an antibiotic
    • only those bacteria that have taken in the plasmid will be able to grow
    • this collection of bacteria is known as a cDNA library
  • Now, the problem is to find bacteria with the specific gene in question 
    • remember, we isolated lots of mRNA from the cells, not just the specific one we wanted
    • the specific gene is found w/ a probe
  • Creation of a probe
    • the amino acid structure of growth hormone is know
    • using the genetic code, they can create a codon sequence that would create this protein
    • they could then create DNA complementary to this sequence - hopefully this sequence (probe) would bind to the DNA
    • the probe is radioactive - this will allow us to identify the bacteria w/ the specific gene in question
  • Isolation of specific colonies of bacteria that have taken in the GH1 gene
    • Lay a filter paper on the colonies - some bacteria will stick to the filter
    • treat filter with chemicals to make double stranded DNA single stranded
    • wash colonies with radioactive probes - these will stick to the DNA that codes for the growth hormone
    • lay filter on film - radioactivity will create exposure, so you know where the gene was
    • look back at the original colony - this colony contains the bacteria w/ the gene
  • Mass Produce the Growth Hormone
    • Transfer cDNA to a new plasmid, one with a bacterial promoter
    • Transform more E. coli w/ the new plasmid - it will now start to produce human growth hormone
    • Isolate and purify the human growth hormone!!

Description of this process

Plasmids - small loops of bacterial DNA

• The main bacterial genome is a large loop of naked DNA - this is where all the "important" genes are stored
• Bacteria also contain smaller loops of DNA called plasmids - supplemental genes are stored there
• Bacteria have the ability to take in foreign DNA and plasmids from the environment (especially when under stress) - this process is known as transformation
  • Bacterial plasma membranes don't normally take in foreign DNA
  • They must be treated w/ chemicals or with electric shock
• Bacteria can also exchange plasmids - this process is known as conjugation

Restriction Endonucleases - enzymes which cut DNA

• Restriction Endonucleases (aka restriction enzymes) cut enzymes at specific DNA sequences
  • Used by bacteria to cut invading viral DNA
  • the sequences of DNA cut are frequently palindromes (the same forwards and backwards - RACECAR)
    • the recognition site (aka restriction site) in bacteria are frequently methylated (have a -CH₃ associated with them) to protect the bacterial DNA from being cut
    • viral DNA does not have methylated restriction sites, so it will be cut by restriction endonucleases
  • Frequently, restriction sites are not cut evenly, but leave "sticky ends," like the enzyme EcoRI

               

  • the sticky ends can be used to insert DNA into plasmids
    • A plasmid with an appropriate restriction site is cut, leaving a long, linear piece of DNA w/ two sticky ends

                  

                    

    • A piece of DNA (typically w/ a gene of interest) is created with sticky ends

                                          

    • The cut plasmid and DNA are mixed - some of the plasmids will join with the DNA, creating a recombinant plasmid

                         

Polymer Chain Reaction (PCR)

A method of replicating DNA millions of times (amplification)

• Processes created by Kary Mullis in the early 80's (sources vary from '83 to '85)

• PCR utilizes Taq polymerase, a DNA polymerase from Thermus aquaticus, a bacterium found in hot springs

  • These bacteria live in hot springs in Yellowstone

  • have DNA polymerase stable at near boiling conditions

• The DNA to be amplified is mixed w/ Taq polymerase and lots of dNTPs (deoxynucleoside triphoshates, the nucleotides used to replicate DNA), and RNA primers

• Denature the DNA - Heating to 94ºC breaks the H-bonds joining the DNA doubles strands, making it single stranded

• Primer Annealing - at cooler temperatures (50-60ºC), the primers anneal to the DNA

• Incubation - Taq polymerase used dNTP's to synthesize complementary DNA strands, starting with the primers

• Repeat 20-30 times to make millions of copies of DNA

Description of this process

Dideoxy Sequencing

A method to sequence DNA

• Process created by Frederick Sanger in 1975 
• Utilizes dideoxyribonucleoside triphosphates (ddNTP)
  • Normal DNA replication adds the new dNTP to the 3'-OH found on the end of the growing chain
  • if a ddNTP is added to the growing chain, there will be no 3'-OH, so no new replication
• When template DNA is mixed with one type of ddNTP (like ddGTP), there will be fragments of different length created

• Do this four times, one for each ddNTP
• Run these on a gel - the gel will separate them based on length
• Read the sequence from the gel

• Recent advances 
  • The Sanger method has be modified - ddNTP's with fluorescent markers have been developed
  • Now we can mix all four types together and do one reaction instead of four
  • The resulting fragments are still of different lengths
  • Separate fragments via electrophoresis in mass-produced, gel-filled capillary tubes. 
  • Automatic sequencing machines read output

RFLP Method of Paternity Testing

RFLP - "Riff-Lips" - Restrictino Fragment Length Polymorphisms

• A RFLP is a pice of DNA that has a "target sequence" surrounded by two restriction enzyme sites

• This can be used to identify DNA left behind in a crime scene - if  RFLP's of the sample will exactly match that of a suspect, the suspect can be convicted (assuming they don't have an identical twin or a clone)

• A Southern Blot can be made to identify these RFLP's

  • Isolate DNA 

  • Cut the DNA with restriction enzymes

  • Run the fragments on an electrophoresis gel

  • Treat DNA with chemicals to make them single stranded

  • Blotting - put a blotting paper on the gel to wick the DNA upwards to a nylon filter, where it is permanently bound

  • Hybridization with a radioactive probe - the complement will bind to the single stranded DNA

  • Autoradiography - place X-ray film over filter - radioactivity will expose film

• For paternity testing, samples are taken from mother, offspring, and potential fathers

• The offspring's DNA is a combination of maternal and paternal DNA - so, all of the offspring's bands should correspond to the mother's, father's or both

  • There is a slight chance of getting unique bands, due to mutation and/or crossing over events

Paternity Testing Demonstration

• Not only is this method useful for paternity testing, but also for genetic markers for diseases
  • In the above picture, the normal - B-globin gene produces three bands, a sickle cell allele produces two bands.
  • By taking samples of blood, the person's genotype can be determined