File - notes

DNA Technology
DNA Cloning
 Recombinant DNA – DNA that comes from two different sources
 Genetic engineering – the direct manipulation of genes for practical purposes
 Biotechnology – the manipulation of organisms to make useful products
 Steps to gene cloning
o Isolate the gene of interest from a eukaryotic cell
o Insert the gene of interest into a previously isolated bacterial plasmid
o The recombinant plasmid is placed in a bacterial cell
o Identify and clone recombinant bacteria
o The gene itself or a gene product can be used for multiple things
 Confer pest resistance to plants
 Alter the metabolism of bacteria
 Produce human proteins
Restriction enzymes – enzymes that cut
DNA molecules at a limited number of
specific locations
o Produce “sticky ends” (single
stranded ends)
o DNA ligase reseals the ends of
the recombinant DNA
o Naming
 The letters refer to the
organism from which
the enzyme was isolated
 First letter – stands
from the genus of the
 Second & third letters –
species name
 Fourth letter (optional
sometimes) – strain of
the organism
 Roman numeral –
whether the enzyme
was first isolated, the
second, etc. from the
E = genus Escherichia
co = species coli
R = strain RY13
I = first RE isolated from this
Recognition sites – sequences of DNA that restriction enzymes
recognize in order to cut the DNA
Cloning vector – a DNA molecule that can carry foreign DNA into a cell
and replicate there
o Plasmid (bacteria & certain fungi)
o YAC (yeast artificial chromosome)
o Virus
Nucleic acid hybridization – process used to identify gene of interest
o Radioactive ssRNA or DNA is used
o Complementary sequence hydrogen binds to the gene of interest
o Requires some knowledge of the sequence
o Requires denaturation of DNA strands of the plasmid
Expression vectors carrying eukaryotic genes often have a
prokaryotic promoter upstream of the gene in order for RNA
polymerase to attach and transcribe the foreign DNA
o DNA that was synthesized from the reverse
transcription of mRNA
o Lacks eukaryotic introns
Yeast (eukaryote!) contains plasmids
o Applying a brief electrical pulse to a solution
containing cells
o Done in order to create temporary holes in the plasma
membrane for foreign DNA to enter
RFLP’s (restriction fragment length polymorphisms)
o Restriction enzymes are used to cut up different
individuals’ genomes in order to compare them
PCR (polymerase chain reaction)
o Quickly amplifies any piece of DNA without using cells
 Target sequence production increases exponentially
o Replicates only target sequences (not whole genome)
o Steps
 Stage 1 (Denaturing)
 Heat briefly to separate DNA strands
 ~ 95°C
 Stage 2 (Annealing)
 Cool to allow primers to hydrogen-bond to DNA
 50-65°C
 Stage 3 (Extending)
 DNA polymerase adds nucleotides to the 3’ end of each primer
 ~ 72°C
o Primers must be present in excess; increases likelihood of attachment to DNA
o Overshoot strands – strands that contain DNA outside the target sequence
o Taq polymerase used in PCR
 Comes from bacteria found in hot springs
 Can withstand high temperatures
 Won’t denature during the denaturing process
o Two primers are necessary per target sequence (one for each DNA strand)
o Overshoot DNA fragments are often not visible if run through gel electrophoresis with the other target
sequence fragments because there are not that many when compared to the number of target sequence
Similarities between PCR and RFLP
o Use gel electrophoresis
o Produce fragments
o Use satellite regions (regions of repetitive DNA)
 Differ between individuals in the number of repetitive
Differences between PCR and RFLP
o PCR – uses only target sequence and primers, doesn’t cut DNA
o RFLP – uses entire genome and restriction enzymes, cuts DNA
DNA Analysis and Genomics
 Genomics – the study of whole sets of genes and their interactions
 Gel electrophoresis – sorts nucleic acids and proteins based on size, electrical charge, and other physical
o Gel used for electrophoresis is a matrix and contains pores that DNA fragments can squeeze through
o The smaller the DNA fragment, the faster it travels through the gel and the further down the gel it appears
o DNA marker contains fragments of known length; a marker must be used with each test to estimate the
length of the fragments run through the gel
Southern blotting combines gel electrophoresis and nucleic acid hybridization to reveal:
o Whether a particular sequence is present in a sample of DNA
o The size of the restriction fragments that contain the sequence
Human Genome Project (HGP)
o Goal: map the entire human genome
o Stages:
 Genetic (linkage) mapping
 The relative distances between gene markers can be determined by recombination
 Physical mapping
 The distances between markers are expressed in some physical measure, typically the
number of nucleotides along the DNA
 A common method for this is known as “chromosome walking”
 DNA sequencing
 The Sanger method uses DNA labeling, DNA synthesis that used special chainterminating nucleotides and high resolution gel electrophoresis
Microarray analysis – used to determine which genes are expressed in particular tissues
In vitro mutagenesis – specific mutation are introduced into the
sequence of a cloned gene and the gene is returned to a cell; the
phenotype of the mutant cell may help reveal the function of the
missing normal protein
SNP’s (single nucleotide polymorphisms)
o Single base-pair variations in the genome
o Occur on average about once in 1,000 base pairs
o Humans are 99.9% related to each other
Prokaryotes – most DNA codes for protein
Eukaryotes - ~97% does not code for protein (nor rRNA, tRNA)
o Regulatory sequences
 Promoter, terminator, enhancers, control elements
o Introns
 Code for snRNPS
o Repetitive DNA
 Positive:
 Telomeres
 Centromeres
 Negative
 Fragile X
 Huntington’s disease
o ncRNA
 miRNA
 siRNA
Gene amplification
o Repetitive sequences of genes increase the genes expression
o Transposition may result in amplification or loss of some genes
 Could increase or decrease transcription
o Transcribed RNA includes the code for an enzyme that catalyzes the insertion of the retrotransposon
(using reverse transcriptase)
Practical applications of DNA technology
o Gene therapy
 Ex) a normal allele is inserted into somatic cells of a tissue affected by a genetic disorder; alleles
has to multiply throughout the patient’s life in order to be permanent (not very effective)
o Pharmaceutical use
 Ex) hormones, vaccines
o Forensics
o Genetically modified organisms (GMOs)
Transformation Lab
 Goals:
o To transform and isolate E.coli bacteria with a previously engineered bacterial plasmid
 Note: the bacterial plasmid has been engineered to contain a eukaryotic gene using the same
process as modeled in the paper plasmid lab
o To identify positive and negative controls designed within the experiment
o To express the eukaryotic gene in the prokaryotic cell
o To consider the practical applications of bacterial transformation in everyday life
The bacterium: E.coli:
o This is a non-pathogenic strain of the bacteria; however, the lab design includes steps to ensure aseptic
technique and to treat the bacterium as if it could cause harm
o The original bacterial colonies used do not have resistance to ampicillin
The pGlo plasmid:
AmpR Gene: This is a gene already within the plasmid that confers resistance to the antibiotic ampicillin
(This not the eukaryotic gene that was introduced)
 The gene codes for the enzymes beta-lactamase that preventsampicillin from entering a bacterial
 Thus, if the bacterial cell contains this plasmid, it will have the AmpR gene and it will be able to
survive in the presence of ampicilin.
o Green Fluorescent Protein (GFP) Gene: This is a gene isolated from a deep sea jellyfish (a eukaryote).
 The gene codes for a green fluorescent protein (this protein fluoresces in black light).
 The gene has been cut out form the jellyfish DNA using restriction enzymes and inserted within
the arabinose operon on the pGlo plasmid.
o Arabinose Operon: This is an operon found within the pGlo plasmid
 The operon normally functions to code for proteins that will breakdown the sugar arabinose
 The plasmid has been engineered to contain the gene for GFP within the arabinose operon.
 Thus, when the operon is turned on (in the presence of arabinose sugar) the GFP gene will also be
expressed and GFP will be produced
Basic procedure
o You are going to take 2 bacterial colonies.
 -pGlo bacteria: These bacteria will NOT be exposed to the plasmid for transformation
 +pGlo bacteria: These bacteria will be exposed to the plasmid for transformation
o For both you will heat shock the bacteria
 The heat makes the membrane more porous and able to accept DNA from the environment (the
 This procedure is NOT very efficient; not all bacteria will be transformed
 But, out of millions of bacteria some should be transformed
o For both, you will add calcium ions.
 The positively charged calcium ions coat the negatively charged bacterial cell membrane and pull
the negatively charged DNA closer to the cell membrane
o You will introduce the bacteria into 4 specific agar plates
 LB-pGlo: This plate contains only Luria Broth and is not exposed to the plasmid
 LB/Amp-pGlo: This plate contains both the Luria Broth nutrients and the antibiotic ampicillin,
and is not exposed to the plasmid
 LB/Amp+pGlo: This plate contains both the Luria Broth nutrients and the antibiotic ampicillin,
and is exposed to the plasmid
 LB/Amp/Ara+pGlo: This plate contains Luria Broth nutrients, the antibiotic ampicillin, the sugar
arabinose and is exposed to the plasmid
Predictions for the plates:
o LB-pGlo:
 Lawn of bacteria
 No ampicillin to kill the bacteria
o LB/Amp+pGlo:
 No arabinose to turn on the
 Some bacteria
 Ampicillin-treated plate
 No glowing because the operon
 Some bacteria absorb the plasmid
is not turned on
 No arabinose to turn on the operon
o LB/Amp-pGlo:
 No glowing because operon is not turned on
 No bacteria
o LB/Amp/Ara+pGlo:
 Ampicillin-treated plate
 Some bacteria
 No plasmid so no ampicillin
 Ampicillin-treated plate
 Some bacteria absorb the plasmid
 No arabinose and no bacteria for
 Arabinose present – operon is turned on
operon to turn on
 Glowing plate because operon is on
 No glowing because the operon
is not turned on/present
o Experimental plate(s):
 This plate shows the effect of our experimental treatment on the bacteria
 LB/Amp+pGlo
 LB/Amp/Ara+pGlo
o Gene Expression Plate:
 This plate shows evidence of our manipulation of gene expression.
 It shows if we have expressed the gene successfully
 LB/Amp/Ara+pGlo
o Positive Control:
 This plate is used to see what bacterial growth should look like before applying our experimental
 If there is no bacterial growth, it indicates that some error has occurred in the lab and the
consequent results may not be reliable
 LB-pGlo
o Negative Control:
 This plate is used to see what no bacterial growth should look like before applying our
experimental treatment/variable.
 If there is bacterial growth, it indicates that some error has occurred in the lab and the consequent
results may not be reliable
 LB/Amp-pGLO
Transformation efficiency
Total number of colonies growing on the agar plate
o Transformation efficiency = Amount of DNA spread on the agar plate (in μg)
Calculate the total number of transformed cells
 Count the number of colonies on the LB/Amp/Ara+pGlo plate
Calculate the amount of plasmid DNA in the bacterial cells spread on the LB/Amp/Ara+pGlo plate
 Calculate the total amount (mass) of plasmid DNA
 DNA in µg = (concentration of DNA in µg/µL) x (volume of DNA in µL)
 Calculate the fraction of plasmid DNA that actually got spread onto the LB/Amp/Ara+pGlo plate
Volume spread on the LB/Amp/Ara+pGlo plate (μL)
 Fraction of DNA used =
Total sample volume in test tube (μL)
Calculate the micrograms of plasmid DNA that you spread on the LB/Amp/Ara+pGlo plate
 DNA spread in µg = (total amount of DNA used in µg) x (fraction of DNA used)
Calculate transformation efficiency using formula
Related flashcards

Peptide hormones

65 cards

Molecular biology

64 cards

Create Flashcards