DNA 4 Gentic Techniques Flashcards
(30 cards)
what does PCR stand for?
Polymerase chain reaction
Describe how PCR works?
Allows specific region of DNA to be amplified from a small amount of sample matter. produces many copies of identical sequence DNA
What are the (3) stages of PCR testing
3 Stages of PCR testing:
- denaturation of double strand DNA (heating it so primers stick)
- Annealing of primers (stick to the parts you want to highlight like flags)
- elongation of strands (multiply copies of identical DNA)
- product=copy of dna (cDNA)
When would PCR be used
Forensic samples archival specimen DNA cloned DNA Geonomic DNA
Wha are the temperatures used in each of the 3 tages of PCR?
- Denaturation of dsDNA - typically temp used is 93-95°C for human genomic DNA. 2. Annealing - temp 40-70°C, depending on melting temperature of the expected duplex. 3. DNA Synthesis - generally 70-75°C: use the optimum temperature for the polymerase being used.
Applications of PCR testing
Diagnosis and screening of genetic diseases and cancer: PCRRFLP, microsatellite, ARMS Rapid detection of slowly growing microorganisms HLA typing in transplantation Analysis of DNA in archival material DNA fingerprinting in forensic science Preparation of nucleic acid probes Clone screening, mapping and subcloning DNA copies from RNA targets RT PCR : Useful for investigating transcription rates, cloning of rare gene transcripts via cDNA libraries and detection of RNA viruses Polymorphism detection : DNA polymorphisms, including all types of mutations and variable number of minisatellite repeats (VNTRs), can be directly detected based upon PCR amplification.
What is RT-PCR testing?
Detection of mRA expression levels. Traditionally involves 2 steps: 1. The RT reaction and PCR amplifications. RNA is first reverse transcribed into cDNA using a reverse transcription as describes here. 2. the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes
Why Analise gene expression?
To find out what genes are switched on in a sample from a particular sample. Use when doing studies/research to compare improvements over time.
What is Genetic engineering
process of mutating genes, usually outside the organisms normal reproductive process.
How is it done Genetic engineering
Involves isolation, manipulation and reintroduction of DNA into cells or model organism usually to express the protein. Examples: Recombinant DNA, cloning and gene therapy
What is Genetic engineering used for?
Recombinant DNA technology is used in creating vaccines, making insulin (Drugs), treatment strategies; research e.g. investigating mechanisms of disease
Outline basic procedure in Genetic engineering
- DNA is purified from cell/tissue 2.Restriction enzymes used to generate specific DNA fragments and cut DNA at specific sequence 3. Fragments join to other DNA molecules which act as vectors (carrier molecules) 4.Recombinant DNA molecule transferred to host cell 5. within host cell recombinant replicatesproducing identical copies (dozens) 6. As host replicates, recombinant molecule passed to all progeny creating population of cells each carrying cloned DNA sequence. 7. Cloned DNA can be recovered from host cells, purified and analysed or 8.cloned DNA in host cells is transcribed, its mRNA translated, gene product isolated and used or sold.
What is cloning
Reproductive cloning: is the creation of an exact genetic replica of a small segment of DNA, a cell or a whole organism. Somatic Cell from organism with desired genes is fused with denucleanated egg cell (nucleous of host egg cell is removed) and this is then encouraged to replicate in tissue culture.
What is cloning used for?
Reproductive cloning only been carried out on sheep/animals:ethically not in humans. Therapeutic cloning of stem cells to help fight disease.
What is Gene therapy
A technique for correcting defective genes responsible for disease development. In most cases a ‘normal’ gene is inserted into the genome to replace an ‘abnormal’ disease causing gene. Although a vector needed to deliver the therapeutic gene to the patients target cells. (lots of worry that introduced virus will combine with natural virus and become harmful to patient). First human gene experiment 1990
Techniques in Gene therapy
Gene Augmentation Therapy and Targeted Modulation of gene expression.
Explain Gene augmentation therapy
A technique used in gene therapy: DNA introduced into a cell with the view to augmenting the gene function eg. recessive diseases. Could be used to deliver genes encoding proteins that would be responsible for stimulating the immune system to treat cancer or infections.
Explain Targeted Modulation of gene expression
A technique used in gene therapy: Methods for turning off genes using for example ribozymes and anti-sense oligonucleotides, eg. infectious disease and cancers
Explain therapeutic cloning and why is is useful
Disease free cells taken from patient. Transfer to denucleated cell egg and cell fused by electrical current Embryo cultured for 7 days Embryonic stem cells recovered Embryonic stem cells grown in culture Specific cells types derived from embryonic stem cells Specific cell types required for therapy reintroduced to patient
What disorders would gene therapy work for?
LoF Diseases. Introduce copies of normal gene which increases the amount of normal product produced in that area. Pathogenises needs to be reversable. (so not dwarfism, as deforminty of gene ocurs at conception) Delivery of therapeutic gene into the cells and preferably into the chromosomes. Targeting of the gene to those cells that require treatment corerct levels of gene expression at the appropriate time, often need to achieve at least 50% expression level. CONCLUSION? Gene therapy not really successful in humans due to the lack of efficient vector systems for gene delivery. However, once this is overcome, cell culture models suggest that DNA delivered to the cells can be expressed and in the case of cystic fibrosis restore function Shift in emphasis to cancer and HIV rather than ‘genetic diseases’
List some uses of Genetic applications
Gene therapy Drugs and vaccines: recombinant proteins Cloning Genetic testing (screening) Forensics – DNA fingerprinting; profiling Genetic screening and counselling Scientific research
Explain DNA testing
1.Sample DNA 2. is cut into small pieces with enzymes that are chemical scissors. 3. DNA feragments are put onto gel 4.fragments are seperated according to size using electrical current (- = largest + = Smallest) 5.Separated fragments transferred to nylon membrane and mixed with DNA probe that picks up the regions of DNA of interest. Needs several probes (why?) To maximise results and help interpret them.
What is Genetic counselling
Provides information and support to an individual or family about genetic disorders
Who uses genetic counselling?
If a child is born with several abnormalities There is history in family of medical condition A woman over 35 is pregnant. A woman has a history of several miscarriages Anyone who has uncertainties about their genetic risks
