8b genome projects and gene technologies Flashcards
what is a genome ?
Entire set of DNA including all the genes in an organism
What are gene sequencing methods?
They work on fragments of DNA which are smaller pieces that are sequenced and put in order to give the whole genome
Why do you want to identify the proteome of DNA?
Useful in identifying medical research and development eg identifying protein antigens on the surface of disease causing bacteria
why do complex organisms contain regulatory genes?
determine when the genes that code for particular proteins should be switched on and off
why do regulatory genes make it harder to translate the genome?
It is harder to translate the genome into the proteome because it is hard to find the bits that code for proteins among non coding and regulatory DNA
What is recombinant DNA technology?
transferring a fragment of DNA from one organism to another
what does the transfer of Dna produce?
a protein in the cells of the recipient of the organism
what is transgenic organisms?
organisms that contain transferred DNA
what are the three ways of producing dna fragments?
method 1 - using reverse transcriptase
method 2- using restriction endonuclease enzymes
method 3 - using a gene machine
How can you use reverse transcriptase ?
- mrna is first isolated from cells
- mixed with free DNA nucleotides and reverse transcriptase
- Reverse transcriptase uses mRNA as a template to synthesis new strands of complementaryDNA
What is palindromic sequences?
Consists of antiparallel base pairs that read the same in opposite directions
what is restriction endonucleases?
enzymes that recognise specific palindromic sequences and cut DNA at places
Why do different restriction endonucleases cut at different specific recognition sequences?
The shape of the recognition sequence is complementary to enzymes active site
What’s the procedure if recognition sequences are present either side of DNA fragment?
- Use restriction endonucleases to separate from rest of DNA
- incubated with specific restriction endonucleases cutting DNA fragment out via hydrolysis
- Cut leaves stickey ends - tails of unpaired bases at each end of fragment
- Binds dna fragment to another piece of dna that has sticky ends with complemtary sequences
How can you use a gene machine?
- the sequence that is required is designed
- First sequence in nucleotide is fixed to some support
- Nucleotides are added step by step in a cycle of processes that include protecting groups
- Oligonucleotides are broekn off from support and protecting groups removed
- Oligonucleotides are joined to make longer DNA fragments.
What is a protecting group?
Make sure nucleotides are joined at the right points to prevent unwanted branching
what is in vivo cloning?
where gene copies are made within a living organism so the dna can be replicated
what is in vitro cloning?
gene copies are made outside of a living organism using polymerase chain reaction
How does part 1 of in vivo cloning work? - recombinant DNA
- Insert DNA fragment into a vectors DNA
- Vector DNA is isolated and restriction endonucleases and DNA ligase are used to stick their dna fragment and vector
- Vector DNA is isolated
- Vector DNA cut open using restriction endonucleases that was used to isolate target gene
- sticky ends of vector complementary to DNA fragment
- Vector DNA and fragment mixed with ligase joining the sticy end of fragment to sticky end of vestor dna - LIGATION
- new combo of bases in DNA
What is part 2 of in vivo cloning? - transforming cells
- Vector cells with recombinant DNA is used to transfer gene into cells
- The host cells take up the vectors containing gene which are then transformed
- A bacteriophage vector will infect host bacterium by injecting its DNA
- The phage DNA integrates into bacterial DNA
What is part 3 - identifying transformed cells?
- Marker genes are inserted into vectors at the same time as the gene to be cloned
- Any transformed host cells will contain the gene to be clones and the marker gene
- Host cells are grown on agr plates and each cell divides replicating dna
- Transformed cells will produce colonies where all cells contain cloned gene and marker
- Marker gene codes for antibiotic resistance
- Only transformed gene with marker will survive and grow
- A marker gene will also calls transformed cells to fluoresce
- Identified transformed cells are allowed to grow more producing lots of copies of the cloned gene
How can you get the transformed host cell to produce proteins?
Make sure that the vector contains specific promotor and
terminator regions
What is in vitro cloning?
Copies of the DNA fragments are made outside organism using a PCR
What is the first step on in vitro cloning?
A reaction mixture containing DNA sample, free nucleotides, primers and dna polymerase is set up
What is primers?
short pieces of DNA that are complementary to the bases at the start of the fragment you want
What is step 2 of in vitro cloning?
The DNA mixture is heated to 95 to break the hydrogen bonds it is then cooled between 50-65 so primers can bind to the strand
What is step 3 of in vitro cloning?
Reaction heated to 72 so DNA polymerase can work and form the new strands
What is step 4 of in vitro cloning?
2 new copies of fragment dna formed and one cycle of pcr complete
What is genetic engineering?
microorganisms, plants and animals can be transformed using recombinant gene technology
How are transformed plants (GM) produced ?
Gene that codes for a desirable protein is turned into a plasmid
Plasmid added to bacterium
Bacterium used as a vector to get the gene into the plant cells
If the right promoter region is added, the transformed cells can produce the desired protein
How are transformed animals (GM) produced ?
A gene that codes for a desirable protein is added into an early embryo or egg cells
The body cells will end up containing the gene
When the female reproduces the cells will contain the gene
What are the benefits of transformed organisms in agriculture?
higher yields
resistance to pests
drought resistant crops
What are the benefits of transformed organisms in industry ?
enzymes produced in large quantities
What are the benefits of transformed organisms in medicine ?
vaccines
drugs
What are the cons of transformed organisms in agriculture ?
might plant only one type of transformed crop making a whole crop vulnerable to the same disease
What are the cons of transformed organisms in industry ?
without proper labelling consumers can be worried about what they have to eat
introduction of toxins
What are the cons of transformed organisms in medicine ?
unethical eg designer babies
what is gene therapy?
altering defective genes inside cells to treat disorders
how does gene therapy work if it is caused by 2 recessive alleles?
add a working dominant allele
how does gene therapy work if it is caused by a mutated dominant allele?
silence dominant gene by sticking a bit of DNA in the middle
what are the 2 types of gene therapy?
Somatic
Germ line
What is somatic therapy?
altering alleles in body cells
What is germ line therapy?
altering the sex alleles so offspring arent affected
What are the ethical issues surrounding gene therapy?
technology could be used for things other than gene therapy
What is a DNA probe?
This is used to locate specific alleles of genes or to see if a persons DNA contains a mutated allele that causes a genetic disorder
How does a DNA probe work?
Short strands of DNA that have a specific base sequence of part of a target allele so the DNA can bind to target alele
Why does a dna probe have a label and what types?
So it can be detected and it will either have a radioactive label or a fluorescent label
How are the fluorescent dna probes used?
- A sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresus
- Seperated DNA fragments are transferred to a nylon membrane and incubated with fluorescent probe
- If an allele is present the dna probe will bind
- the membrane is then exposed to UV light and if a gene is present there will be a fluorescent band
what is a DNA microarray?
a glass slide with microscopic spots of different DNA probes attached to it in rows
How does the DNA microarray work?
- Fluorescent DNA washed over array
- If the labelled DNA contains DNA sequences that match the probes it will stick to the array so the DNA vna be screened for the mutated genes
- Array is washed to remove any fluorescently labelled DNA that hasn’t stuck and then visualised under uv light
- Any spot that fluorescents means the persons DNA contains that specific allele
What are the uses of screening with DNA probes?
identify inherited conditions
determine how a patient responds to specific drugs
identify health risks
What is genetic counselling?
Advising patients and relatives about genetic disorders risks and whether they are a carrier, the type they have and the treatment
What is personalised medicine?
genes determine how you respond to specific drugs so we can tailor medicine to a persons dna
what is genetic fingerprinting?
the number of times a sequence is repeated at different places in the genome can be compared between indivudals
who uses genetic fingerprinting?
forensic scientists to identify people by their dna
How do you produce a genetic fingerprint? What is step 1?
PCR is used to make DNA fragments
- DNA obtained
- Copies of area that contain base sequences that do not code are made
- primers bind to these repeats
- Dna fragments where the length in nucleotide corresponds to number of repeats a person has at a specific position
- A fluorescent tag is added to dna so tehy can be viewed under UV
What is step 2 in genetic fingerprinting?
Separation of DNA fragments by gel electrophoresis
- DNA mixture is placed into a well in a slab of gel and buffer solution that conducts electricity
- Electrical current is passed through the gel - DNA fragments (-ve) charged so move towards (+ve) charged
- Pattern of bands
how do dna fragments separate?
according to length so shorter travel further through the gel
What is step 3 in genetic fingerprinting?
- gel placed under uv light
- dna fragments seen as bands making up a fingerprint
how do you compare the fingerprints?
if the band is at the same location on the gel they have the same number of nucleotides so same number of non coding repeats
What is the uses of genetic fingerprinting?
- determining genetic relationships
- determining genetic variability
- forensics science
- medical diagnosis
- animal and plant breeding
what is a vntr?
variable number tandem repeats that dont code for proteins
