Lectures 8-9 Outline: Recombinant DNA, Biotechnology, and Human

Transcription

Lectures 8-9 Outline: Recombinant DNA, Biotechnology, and Human
Lectures 8-9 Outline: Recombinant DNA, Biotechnology, and Human Disease
1) List techniques that are used by molecular biologists to investigate and diagnose cellular and
disease-state processes.
a) Cloning DNA (Recombinant DNA)
i) Artificially produced sequences of DNA that are formed from fragments of nucleic acid not
normally found in nature.
ii) Cloning is the process of recombining DNA from one source (e.g., specific human DNA)
into another location so that it can be maintained.
iii) Typically, this occurs by moving the source DNA into a plasmid that can replicate in a
bacterial host.
iv) Use enzymes called restriction endonucleases to cleave short sequences of DNA (4-6 bp)
and cleave phosphodiester bond in both DNA strands
(1) RE sites are palindromes
b) Polymerase Chain Reaction
c) Nucleic Acid Electrophoresis
d) Sequencing
e) DNA Hybridization
(1) Uses Include:
(a) Southern Blotting (detecting DNA)
(b) Northern Blotting (detecting RNA)
(c) Genetic Disease Diagnosis
(d) Pathogen Identification
(e) Microarrays (assaying gene expression)
f) Transgenic animals
g) Restriction Fragment Length Polymorphism
h) Fingerprinting
2) Describe the most common enzymes employed in these techniques.
a) Restriction Endonucleases—cut double stranded DNA at specific sequences. Used in cloning
and some diagnostic assays (e.g., RFLP).
b) DNA Polymerase—polymerizes a new DNA molecule that is complementary to an existing
template strand. It has many uses in molecular biology.
c) Heat stable DNA polymerases—a class of DNA polymerases, isolated from thermophilic
bacteria, that remain active at high temperatures. Taq DNA polymerase (from Thermus
aquaticus) is the most common example. Uses include PCR and cycle sequencing.
d) DNA Ligase—forms a phosphodiester linkage between the 5’-phosphate and the 3’-hydroxyl
of adjacent nucleosides on “nicked” DNA. Used in cloning.
e) RNA Polymerase—polymerizes an RNA molecule that is complementary to an existing DNA
template.
f) Reverse Transcriptase—polymerizes a DNA molecule that is complementary to an RNA
template. It is used in the production of cDNA and to identify transcriptional start-sites of
genes.
3) Describe how genetic elements can be cloned, and what host cells may be used.
a) See explanation under discussion of restriction endonucleases
4) Explain what PCR is, and how it works.
a) The polymerase chain reaction is used to amplify sequences from a small sample of DNA
b) PCR can increase the portion of a particular DNA sequence in a mixed DNA population
5) Describe two fundamentally different technologies used to sequence DNA.
a) Base specific chemical cleavage
b) Di-deoxynucleic acid incorporation
i) Based on absence of a 3’ hydroxyl group
ii) Incorporation of a dd nucleotide means no place for additional 5’ phosphates to be added
iii) Termination of polymerization
iv) Three types:
(1) Sanger sequencing using radioactive substrates
(a) Figure 1. The Sanger sequencing reaction. Single stranded DNA is amplified in the
presence of fluorescently labelled ddNTPs that serve to terminate the reaction and
label all the fragments of DNA produced.
The fragments of DNA are then separated
via polyacrylamide gel electrophoresis
and the sequence read using a laser beam
and computer.
(2)
(3)
(4) Use of fluorescent markers
(5) Cycle sequencing
6) Define gel electrophoresis.
a) Gel is formed in a cast. It may be made of polyacrylamide or agarose
b) Samples are applied to the wells. Samples contain glycerol so that they sink into the well and a
dye so samples can be seen
c) An electric current is applied across the gel, causing the migration of charged molecules in the
sample
d) Separation occurs according to size for molecules (like nucleic acids) with a constant charge to
weight ratio. Visualization of DNA usually requires additional steps such as staining or
autoradiography
e) Stuff migrates from wells at negatively charged end, dye front at positively charged end
7) Describe how DNA hybridization is used to identify specific alleles.
a) How do we know where probe is binding?
i) Oligonucleotide probes can be (usually) covalently linked to a reporter that makes them
easy to detect in an assay.
ii) Radionuclides
iii) Fluorophores
iv) Enzymes with colorometric products
v) Biotin-Avidin interaction
b) Can use allele-specific oligonucleotides (ASO)
c)
8) Give an example of how hybridization techniques can be used in medical diagnosis.
i) The probe is an oligonucleotide (typically synthetic).
ii) The label is may be radioactive, fluorescent, or enzymatic.
iii) Hybrid molecules indicate sequence similarity between probe and the nucleic acid to
which it is bound.
b) Diagnosis of Sickle Cell Disease using RFLP (not the same as using ASOs)
i) Changes in restriction endonuclease cleavage patterns (RFLPs) can be detected by cutting
chromosomal DNA with the appropriate enzyme, and using Southern hybridization to
identify the affected fragments
c) Example of nucleic acid hybridization to detect bacterial pathogens
i) Detection of chlamydial DNA by direct hybridization.
ii) The Gen-Probe Pace 2® DNA hybridization test.
iii) All nucleic acid-based chlamydial diagnostic assays depend on hybridization. In direct
hybridization probe tests, signal is generated without any intermediary target
amplification.
iv) The Gen-Probe PACE 2 test was first introduced in 1988. Its sensitivity is founded on the
fact that it is targeted against high copy number chlamydial rRNA (16S) which effectively
serves as a natural pre-amplification.
v) As rRNA is mostly single stranded, no thermal denaturation for strand separation is
necessary prior to hybridization, which takes 60 minutes at 60C.
vi) The test is completed in approximately two hours (Fig 1). The test is popular because of its
relative simplicity to perform and its scalability using conventional laboratory equipment.
An acridinium-ester probe generates a signal in the LEADER® luminometer following
addition of hydrogen peroxide.
d) Example of Nucleic Acid Hybridization to Detect Viral Infections
i) Human Papillomavirus: primary agent in cervical cancer, 2nd most common type of cancer
in women worldwide. 3rd leading cause of cancer-related deaths in women worldwide.
Most common viral STI that goes undiagnosed due to no symptoms developed
ii) HPV types
(1) Types 6 and 11 – most common low-risk HPV
(a) Associated with genital warts and rarely found in cervical cancer
(2) Types 16 and 18 – most common high-risk HPV
(a) Associated with cancers of cervix, vagina, vulva, anus, and penis
9) Define “Northern blotting.”
Northern blotting uses MRNA as the target for probes
10) Explain gene therapy.
a) Mutated or dysfunctional genes for a variety of diseases have been determined
i) Ex: huntington’s, cystic fibrosis, Duchenne’s muscular dystrophy
b) Augmentation of replacement of such faulty genes may be possible
c) Retroviruses as vectors for delivery of gene therapy
i)
Genes encoding human proteins may be cloned into
defective retroviruses
ii)
Such viruses are missing essential genes for active infection,
but can enter host cells to deliver genes that may be incorporated into the
host genome
d) The goal of gene therapy is to insert the normal cloned DNA for a
gene into the somatic cells of a patient who has a defect in that gene as a
result of a mutation
e) Patients with severe combined immunodeficiency disease (SCID)
have an immune deficiency as a result of mutations in either the gene for
adenosine deaminase or a gene coding interleukin receptor subunit.
f) Both diseases have been corrected using gene replacement therapy
g) Side effects – Retrovirus-mediated gene transfer was able to
correct SCID-X1 in nine of ten patients. However, leukemias developed in
several of the patients, possibly through activation of a hematopoietic
oncogene
h) Factors affecting gene therapy
i)
Gene vector system
(1) Predictability of integration at acceptable loci
(2) Controllable infectivity
(3) Host immune response
ii)
Regulation of replacement gene expression
(1) Spatially (in correct cells)
(2) Temporally (at the correct time)
(3) Expression strength (in the correct amount
iii)
Multigene disorders
11) Explain how a restriction endonuclease can be used in the analysis of patient samples to
determine the presence or absence of a particular genetic locus.
a) Restriction fragments are “sticky” and “blunt”
b) Restriction enzymes cleave dsDNA and produce a 3’-hydroxyl group on
one end and a 5’-phosphate group on the other
c) For example, TaqI forms a staggered cut that produces sticky or
cohesive ends (5’-overhang)
d) Conversely, HaeIII produce fragments that have “blunt” ends that are
double stranded
e) Steps for DNA Cloning
i) Introduction of a foreign DNA molecule into a replicating cell
permits cloning or amplification of that DNA.
ii) To clone a DNA fragment or nucleotide sequence of
interest, total cellular DNA is cleaved with a specific
restriction nuclease, creating thousands of fragments
iii) Each fragment is cloned (ligated with a ligase) into a
cloning DNA vector
iv) The vector is a molecule of DNA to which the fragment
of DNA to be cloned is joined
v) Essential properties of a vector include:
(1) Autonomous replication in host cell
(2) Contain at least one specific nucleotide sequence
recognized by a restriction endonuclease
(3) Carries at least one gene that confers the ability to
select for the vector, such as an antibiotic resistance
gene
vi) Commonly used vectors: plasmids or viruses
Overview of steps in cloning
vii) Sources of DNA for cloning
(1) Restriction digests of DNA from various sources
(2) cDNA libraries
(3) PCR products
AAAAAAA
Selecting the Right Clone
TTTTTT
T
AAAAAA
A
mRNA
5’
5’
mRNA
Anneal
primers,
AAAA
AAA
TTTTTTT
TTTTTT
Use
RNase
mRNA
DNA
5’
3’
viii) Hosts for Recombinant Gene Expression
(1) Bacteria: relatively simple expression systems, E. coli is most common
(2) Yeast: can carry out some post-translational modifications, YACs
(3) Humans (gene therapy)
(4) Transgenic animals: mammalian hosts (expression in milk), high yields and potential
for progeny expression
(a) Ex: tissue plasminogen activator (TPA) is an endopeptidase synthesized by
endothelial cells that binds to fibrin clots and catalyzes the cleavage of
plasminogen to the active form of plasmin. t-PA produced by recombination
technology (using goat as host) is used for therapeutic thrombolysis
12) Give an example of a role that reverse transcriptase may have in the therapy of a metabolic
disorder.
13) Describe how viruses can be used in the treatment of genetic disorders.
a) Recombinant vaccines can be made
i) The general idea is that injection of the recombinant vaccinia viruses into patients will
result in the production of viral proteins
ii) It is anticipated that an immune response will initiate against these proteins and impart
immunity to these infections
iii) Currently, recombinant DNA vaccines are expressed in other organisms and recombinant
viral proteins purified
iv) These purified proteins are then used as antigens for antibody production
v) Ex: GARDASIL
14) Describe how the analysis of VNTR’s can lead to the identification of specific individuals.
a) Single nucleotide polymorphisms (SNPS) constitute about 90% of differences between human
genomes
b) There can also be polymorphisms where variable numbers of tandem repeats (VNTR) occur. A
specific number of tandem repeats defines a VNTR at a particular locus (ex: different numbers
of GC’s in different individuals)
c) DNA fingerprinting: differences in sequences result in altered endonuclease cleavage sites.
These differences offer “genetic fingerprints” by which individuals can be distinguished
d) VNTRs can be detected by PCR (amplify them and then electrophorese on gel)
15) Explicate the use of PCR in diagnose of viral disease. Contrast this method of diagnosis with the
use of Western blot analysis to diagnose the same viral disease.
16) Describe what a gene chip is and outline how they could be used to test for the possibility of
adverse reaction to potential drugs for an individual patient.
a) Microarrays (DNA chips) are small glass slides onto which 1000’s of individual sequences can
be spotted
b) DNA from patients can be labeled and used to probe the sequences on the chip
c) This technology can be applied in a variety of ways to answer questions about the patients
genome sequences
d) They can also do the following
i) Identify which mutation, among several, is present in a patient
ii) Identify which alleles of drug-metabolizing genes are present in a patient
iii) Screen for the presence of pathogen DNA
17) Using PCR to test genetic fitness and identify genetic disorders
a) PCR greatly reduces the time required to diagnose genetic disease
b) No cell culture is needed
c) Genetic disease that require fetal development before they are evident can be detected
earlier
d) DNA can be obtained from amniotic fluid or chorionic villi
18) Use of RFLPs analysis for diagnosis of phenylketonuria
a) You can apply restriction enzyme cleavage and southern blotting to identify the disease
producing gene
b) The presence of abnormal genes for PAH can be shown using DNA polymorphisms as markers
to identify normal and mutant genes
c) The presence of a polymorphic site creates fragement b and is associated with the mutant
gene
d) The gene for PAH is deficient in those with phenylketonuria or PKU
e) The gene is on chromosome 12 and spans about 90 kb and contains 13 exons separated by
introns
f) Mutations in the PAH gene usually do not directly affect any restriction endonuclease site
g) For a diagnostic protocol on must look at the DNA of family members and identify genetic
markers (RFLPs) tightly linked to the disease trait
19) Small, Interfering RNA
a) Involved in RNA interference pathway where siRNA interferes with the expression of a
specific gene
DNADNA
TTTTTTT
5’ 3’
cDNA
Constructi
on
b) RNAi silencing is initiated when double-stranded RNA (dsRNA) is processed into small
interfering RNAs (siRNA) between 19-26 base pairs in length by an RNaseIII enzyme called
Dicer.
c) These siRNAs are subsequently incorporated into RNA-induced silencing complexes (RISC)
that target cognate messenger RNA (mRNA) for cleavage to mediate gene suppression
d) This represents a way to reduce expression of a specific mRNA
e) This transcript-specific expression can occur through either specific degradation of the mRNA
or blocking translation of the transcript.
f)
20) Micro RNA and Gene Silencing
a) Eukaryotic cells have a built-in
system to silence gene
expression via reduction in
mRNA levels – gene silencing
b) Gene silencing comes via
transcription of genes known as
microRNAs (miRNA)
c) miRNA genes are transcribed in
the nucleus by RNA polymerase
II, generating primary miRNA
transcripts, process to premiRNA and then exported to the
cytoplasm.
d) In the cytoplasm, pre-miRNA is further processed by a ribonuclease (Dicer) and the resulting
double stranded miRNA is strand selected with the guide strand (black strand) entering the
RISC (RNA-induced silencing complex)
e) The guide strand of RISC targets the complex of the 3’-untranslated region of the target
mRNA, leading to degradation of the mRNA or translation inhibition

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