DNA Techniques Flashcards
How did the somatic cell nuclear transfer work for Dolly the Sheep?
Nucleus from mammary micro injected into enucleated unfertilized oocyte, zygote transferred to pseudopregnant female, exact genetic copy of the nuclear donor animal developed
Define clone and give examples.
Simply an exact genetic copy
Bacteria are clones since they reproduce by binary fission
Dolly the sheep is a clone of her “mother”
What is recombinant DNA?
Refers to joining of a DNA sequence of interest to DNA originating from another source (plasmid of bacteriophage)
What does recombinant DNA allow?
This allows the isolation and amplification of a specific region of chromosomal DNA (cloning); once amplified, detailed analyses of the DNA structure/protein function are possible
What is the basis for most recombinant DNA techniques?
Complementary base pairing
What are some sources for DNA for cloning?
Whole genomic DNA
Chromosome specific DNA
cDNA/tissue specific cDNA
How is cDNA synthesized?
Using a retro viral enzyme (reverse transcriptase) to make DNA copies of mRNA.
What are vectors and what are some examples of vectors?
They are molecules that can replicate autonomously in a host.
Some examples include: Plasmids Bacteriophage Cosmids Yeast artificial chromosomes (YAC)
What does the lyric cycle do to the host?
Kills it
What does the lysogenic cycle result in?
Production of dormant prophage
What are some bacterial enzymes used in cloning?
Type II restriction endonucleases
DNA ligase
What do type II restriction endonucleases do?
They are a bacterial defense mechanism
They recognize 4-8 bp palindromes and cleave them often producing cohesive “sticky” ends
What happens when a plasmid is introduced to a new host
Transformation
What happens when a bacteriophage is introduced to a host?
Transduction
What is transformation?
A natural mechanism of bacterial genetic transfer
What is transduction?
Infect bacteria with recombinant virus
What happens in phage lambda cloning?
Genes for viral integration into host genome (lysogenic life cycle) are removed and replaced by foreign DNA
What do recombinant phases from phage cloning do?
They are infective and will form plaques
What are some commonly used probes in probe design?
Cloned DNA fragment from another species
Antibody specific for gene product
PCR amplified DNA fragment
Synthesized DNA fragment based on protein sequence
What does sequencing DNA tell you?
Determines protein sequence which can give clues about protein function
What do we use expressed recombinant proteins for?
It’s how we get things like insulin, human growth hormone and clotting factors
Can you use cloned DNA as a probe?
Yes, it can be used in the detection of mutations in genes (prenatal)
Uses as minisatellite sequences to produce DNA fingerprints (forensics)
How can cloned DNA be used in functional studies?
Detect mRNA expression of the gene in tissues, individual cells, in response to signaling molecules
How can cloned DNA be used in gene therapy?
Transfer it into the genome to correct a genetic defect
What is one method of DNA sequencing?
Sanger Dideoxy (ddNTP) Method
How does the Sanger Dideoxy Method work?
DdNTP analogs inhibit DNA polymerase as it synthesizes the complementary strand
How was the genome project conducted?
Fully automatically at 1000 bases/sec
What is subcloning?
Once isolated, a cloned gene can be excised from the cloning vector and transferred to a different, more specialized vector
What can subclones be used for?
Site-directed mutagenesis
Fusion protein production
In vitro translation
Production of recombinant proteins
What is site directed mutagenesis used for?
Used to map functional domains of the gene products
How do expression vectors work?
Specialized plasmids are used to express proteins in bacteria. This is where we get things like insulin and hGH.
What is green fluorescent protein used for?
It is a fusion protein used to tag proteins to monitor when and where they are expressed in an organism
What is southern blotting used for and how does it work?
It is used for forensic analysis.
Repetitive element probes are used to identify individual patterns
What is RFLP analysis used for?
Prenatal diagnosis using the concept of linkage
What is microarray technology useful for?
Comparing genes expressed in 2 populations of cells or tissues
What is siRNA useful for?
For knocking down protein levels and monitoring effect on cell behavior
What is polymerase chain reaction (PCR) useful for?
Making copies for further analysis by allowing selective amplification of a single molecule of DNA several million fold in just a few hours
Does the human genome project support use of PCR for gene cloning?
Yup
How many DNA regions are tested and stored in CODIS for forensic analysis.
13 matches
What are some challenges for proper expression in gene transfer?
Vectors must contain eukaryotic elements
Gene must be integrated into host chromosome
Site of integration is mostly random
What is a knockout?
If introduce inactive gene and homologous lay recombine it into correct site in genome.
What is “pharming”?
The term used for introducing genes for human proteins or pharmaceuticals into traditional farm animals and harvesting the products
What is the most reliable form of gene therapy and how does it work?
Ex vivo: performed on cells that can be removed from the patient, manipulated and returned.
How does in vivo gene therapy work?
Corrected gene is injected into patient via disabled retro viral vectors for the most part
What are some non-viral delivery systems for gene delivery?
Liposomes
Non-lipid coats
Naked DNA
Mini chromosomes
Is it legal to perform gene therapy on germ cells or embryos?
Nope
What optometry related disease has gene therapy been used to treat?
Choroideremia
How has gene therapy been used in dentistry?
Bone regeneration Periodontal ligament regeneration Dentin regeneration Salivary gland regeneration Oral cancer treatment
Describe totipotent stem cells.
From zygote
Can form whole human being
Describe pluripotent stem cells.
From blastocyst
Can form cell types from all 3 germ layers
Describe multipotent stem cells.
From adult
Can form many closely related cell types
