BIOL 1001 Study Guide - Final Guide: Dna Ligase, Recombinant Dna, Genetically Modified Bacterium
9 Jun 2018
School
Department
Course
Professor
Biotechnology
●Applications of DNA:
○Classic biotechnology- fermentation, cheese production, wine, yogurt, beer,
production etc.
○Recombinant DNA technology uses enzymes to splice and form new DNA
molecules not normally found in nature
■Splicing genes from one organism into another (produces a transgenic
organism) trans means “across”
■Enzymes needed: Rest, Enzymes, DNA ligase, DNA polymerases,
Reverse transcriptase, etc.
Making Recombinant Data
1. Restriction Enzymes- discovered in the late 1960s
a. Restriction enzymes cut double-stranded DNA into fragments not RNA, cleave
bonds in DNA backbone (sugar-phosphate)
b. Also known as restriction endonucleases (do not cut at ends of a DNA molecule)
c. Used in nature to ward off attack of bacterial viruses
d. These enzymes do not end in the suffix -ase
e. Fragments base-pair at sticky ends
f. Restriction enzymes recognize palindromic sequences in the DNA (also known as
inverted repeats- racecar, bob, dad
g. Some not all give rise to “sticky ends” these are the ss DNA strands
complementary to other ss strands (complementary base pairing)
h. Other restriction enzymes give rise to blunt ends
i. Remember that two strands of DNA run in opposite directions
2. DNA Ligase Enzyme- seals up breaks in both strands of the DNA- phosphodiester bonds
3. Plasmids- developed as cloning vectors
Using Plasmids
●A plasmid is a small circle of bacterial DNA outside the chromosomes
●Plasmids in bacteria give rise to microbial resistance to antibiotics
●Foreign DNA can be inserted into a plasmid
○Forms recombinant plasmids
○Plasmid is a cloning vector
○Can be used to deliver DNA into another cell, or cell of another organism
○Naturally occuring plasmids can be engineered to produce a plasmid cloning
vector
●Plasmid insertion
○Cut plasmid with a restriction enzyme such as EcoRI
○Cut foreign DNA with same rest enzyme
○Add cut DNA to the linearized plasmid
○Zip up plasmid and insert with DNA ligase enzyme
Early Cloning Experiment
●Used ribosomal RNA genes
●Agarose gel stained with ethidium bromide
1. Technology: Polymerase Chain Reaction
○DNA sequence should be known sequence to be copie is heated to melt into single
strands
○Single-stranded DNA primers added, and bind to ends of both single strands of
template DNA
○DNA polymerase uses free nucleotides to create complementary strands
○A special DNA polymerase is used
○This polymerase is heat stable, Taq DNA Polymerase
○Doubles number of copies of DNA every cycle
○PCR (1)
i. Double stranded DNA to copy
ii. DNA heated to 90-94C, primers added to base-pair with ends
iii. Mixtures cooled’ based-pairing of primers and ends of DNA strands
iv. DNA polymerases assemble new DNA strands
○PCR (2)
i. Mixture heated again; makes all DNA fragments unwind
ii. Mixture cooled; base-pairing between primers and end of single DNA
strands
iii. DNA polymerase action again doubles number of identical DNA
fragments
Enzyme for PCR
●The enzyme for PCR is called Taq DNA polymerase
●From thermophilic organism thermus aquaticus
, which lives in hot springs. This
bacterium thrives at temperatures around 70 degrees Celsius
● Taq DNA polymerase
is stable at higher temperatures
II. Traditional DNA Sequencing
●How can you find out the DNA sequence of a gene?
●Once you kno the DNA sequence, can develop a genetic test- Huntington’s disease, PKU
etc.
●Nucleotides are assembled to create complementary strands
●When a modified nucleotide is included, DNA synthesis stops
●This is known as the dideoxy method
●Result is millions of tagged copies of varying length
DNA Sequencing
●Ultimate goal of mapping is determining nucleotide sequences for each chromosomes
●Steps of approach:
○Cut DNA from many copies of chromosomes in to overlapping fragments
○Clone fragments in plasmid of phage vectors
○Sequence fragments
○Order sequence into overall sequence
B. Next Gen DNA Sequencing
●Next gen sequencing (high throughput sequencing) allows rapid and repeated
sequencing of human DNA by parallelizing the sequencing process
●Produce thousands or millions of DNA sequences at once
●Will soon be possible to sequence a single person's DNA for approximately $1000 in one
to two days
●Will have huge ramifications for human health and medical science
○Ex: Apo e-4 allele AD
●BRCA1 gene predicts breast and ovarian cancer
○Would allow preventative medicine to be used (pharmacogenomics)
○Correlate specific genes with medical histories
●Analytic skill and knowledge needed to make use of genome info (3 trillion bases) is
impt.
○Challenge is to deliver value of DNA sequence data out to physicians
Deep Sequencing
●Technology allows a DNA fragments to be repeatedly sequenced in a very short time
●Sequencing is done in a parallel fashion. Sequencing by synthesis approach
C. Personalized Medicine
●If DNA sequence of a gene (or genome) is known- may oredict efficacy (usefulness) of a
drug
●Apo34 and adult onset Alzheimer’s Disease- gene differs at two SNP sites
●BRCA1, BRCA2- BC
●PHARMACOGENOMICS
●Herceptin is an antibody
●HER2-rec positive and HER2
●Reg negative breast cancer
Human Genome Project and DNA Sequencing
●Also known as HUGO
●Goal- map the entire human genome- all twenty four chromosomes- 22 autosomes, X and
Y chromosomes
○Used technique known as dideoxy sequencing
○Initially thought by many to be a waste of resources
○James watson was the first director and was replaced by F. Collins
○Processed accelerated when Craig Venter used bits of cDNAs as hooks to find
genes (shotgun technique)
○Genome sequencing was completed ahead of schedule in early 2001 by a private
company ( Celera) and by NIH
○OFFICIALLY SEQUENCE IN 2003
Surprising Discoveries from HUGO
Document Summary
Classic biotechnology- fermentation, cheese production, wine, yogurt, beer, Recombinant dna technology uses enzymes to splice and form new dna. Splicing genes from one organism into another (produces a transgenic. Enzymes needed: rest, enzymes, dna ligase, dna polymerases, molecules not normally found in nature organism ) trans means across . A plasmid is a small circle of bacterial dna outside the chromosomes. Plasmids in bacteria give rise to microbial resistance to antibiotics. Foreign dna can be inserted into a plasmid. Can be used to deliver dna into another cell, or cell of another organism. Naturally occuring plasmids can be engineered to produce a plasmid cloning vector. Cut plasmid with a restriction enzyme such as ecori. Cut foreign dna with same rest enzyme. Add cut dna to the linearized plasmid. Zip up plasmid and insert with dna ligase enzyme. Agarose gel stained with ethidium bromide: technology: polymerase chain reaction.
