DNA 4 Gentic Techniques

Question 1

what does PCR stand for?

Answer 1

Polymerase chain reaction

Question 2

Describe how PCR works?

Answer 2

Allows specific region of DNA to be amplified from a small amount of sample matter. produces many copies of identical sequence DNA

Question 3

What are the (3) stages of PCR testing

Answer 3

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)

Question 4

When would PCR be used

Answer 4

Forensic samples archival specimen DNA cloned DNA Geonomic DNA

Question 5

Wha are the temperatures used in each of the 3 tages of PCR?

Answer 5

1. 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.

Question 6

Applications of PCR testing

Answer 6

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.

Question 7

What is RT-PCR testing?

Answer 7

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

Question 8

Why Analise gene expression?

Answer 8

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.

Question 9

What is Genetic engineering

Answer 9

process of mutating genes, usually outside the organisms normal reproductive process.

Question 10

How is it done Genetic engineering

Answer 10

Involves isolation, manipulation and reintroduction of DNA into cells or model organism usually to express the protein. Examples: Recombinant DNA, cloning and gene therapy

Question 11

What is Genetic engineering used for?

Answer 11

Recombinant DNA technology is used in creating vaccines, making insulin (Drugs), treatment strategies; research e.g. investigating mechanisms of disease

Question 12

Outline basic procedure in Genetic engineering

Answer 12

1. 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.

Question 13

What is cloning

Answer 13

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.

Question 14

What is cloning used for?

Answer 14

Reproductive cloning only been carried out on sheep/animals:ethically not in humans. Therapeutic cloning of stem cells to help fight disease.

Question 15

What is Gene therapy

Answer 15

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

Question 16

Techniques in Gene therapy

Answer 16

Gene Augmentation Therapy and Targeted Modulation of gene expression.

Question 17

Explain Gene augmentation therapy

Answer 17

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.

Question 18

Explain Targeted Modulation of gene expression

Answer 18

A technique used in gene therapy: Methods for turning off genes using for example ribozymes and anti-sense oligonucleotides, eg. infectious disease and cancers

Question 19

Explain therapeutic cloning and why is is useful

Answer 19

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

Question 20

What disorders would gene therapy work for?

Answer 20

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’

Question 21

List some uses of Genetic applications

Answer 21

Gene therapy  Drugs and vaccines: recombinant proteins  Cloning  Genetic testing (screening)  Forensics – DNA fingerprinting; profiling  Genetic screening and counselling  Scientific research

Question 22

Explain DNA testing

Answer 22

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.

Question 23

What is Genetic counselling

Answer 23

Provides information and support to an individual or family about genetic disorders

Question 24

Who uses genetic counselling?

Answer 24

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

Question 25

Genetic screening and counselling

Answer 25

Carried out to see if genetic testing needs to be done

Question 26

Explain Genetic Testing

Answer 26

It is the examination of a persons chromosomes, DNA or the biochemical product of a gene (protein)

Question 27

List somes tissues that would be taken in Genetic testing.

Answer 27

Blood Skin Saliva Hair follicles ammniotic fluid CVS (11-12 weeks, most risky) Placental (15-19 weeks, less risk to baby)

Question 28

When can Genetic testing in and around pregnancy be done and list some examples of tissues/samples taken

Answer 28

Can be done pre-pregnancy (blood test); in pregnancy (e.g, CVS, amniocentesis, blood test); after pregnancy (e.g guthrie test, blood test in adult)

Question 29

List the Types of genetic tests used.

Answer 29

Cytogenic testing - examine chromosomes (read chromosomes chart) DNA testing – DNA extracted from cells of a variety of body fluids or tissues Proteins – usually tested for in blood

Question 30

RESEARCH: Diseases and Genetic techniques:

Answer 30

Genetic techniques used all the time to investigate diseases Identify therapeutic and diagnostic markers Understand molecular mechanisms involved in development of diseases Identify genes that are abnormal and cause disease