Genome Projects + DNA Technology Flashcards
What is the human genome project
A project which successfully determined the sequence of bases of a human genome
= aim to sequence entire human Genome
What is the genome
Complete set of genes present in an organism
How is the human genome project used in medical advances
Genome projects identify which genes are responsible for certain inherited diseases = once genes are determined potential targets for drug treatment can be identified
What is genetic testing/screening
Used to identify if an individual has a specific gene that may cause a disease
Done with DNA probes complimentary to genes of diseases
What is gene therapy
Used to replace defective genes with normal,healthy genes
How can genome projects be used in evolutionary relationships
Make comparisons between individuals or species = genetic similarities can help us to show evolutionary relationships
- can also be used in forensic testing + genetic
Why have we sequenced the genome of other organisms such as zebra fish
To identify useful genes = may help us with our own problems
Recombinant DNA technology
Transfer of DNA fragments from one organism to another
Why can transferred fragments in Recombinat DNA technology be translated within recipient organism
DNA is universal
Name 3 ways fragments can be produced
Reverse transcriptase
Restriction endonuclease
Gene machine
What happened after fragments are produced
They need to be amplified
Either by :
1- in Vitro
2- in Vivo
(Vinland saga)
Producing fragments - Reverse transcriptase
mRNA converted to DNA by enzyme Reverse transcriptase = produces cDNA (complimentary DNA)
mRNA is more accessible than DNA + there is usually a lot of it within a cell
Producing fragments - Restriction endonucleases
Restriction endonucleases cut DNA at specific base sequences known as RECOGNITION SITES = allows desired gene to be cut out of DNA and isolated
What 2 types of fragments can restriction endonuclease produce
Blunt end = cut at the same location = no exposed bases
Sticky end = staggered ends with exposed DNA bases
- the exposed ends are Palindromic and read the same forwards on one end and backwards on another
Producing fragments - Gene machine
DNA sequence entered into a computer = computer synthesis DNA strand
Procedure of the gene machine
1- If we know the desired protein amino acid sequence = work out RNA and DNA sequence
2- info fed into computer = computer checks sequence for safety to make sure we don’t produce DNA for pathogen
3- computer then forms desired gene from series of Oligonucleotides (single strands of nucleotides)
4- PCR used to amplify + make double stranded
One advantage and disadvantage of reverse transcriptase
+ mRNA in cell is from the desired gene and there is lots of it
- more steps = more time consuming
One advantage and disadvantage of restriction endonucleases
+ sticky ends on DNA fragment makes it easier to insert to make recombinant DNA
- may contain introns
One advantage and one disadvantage of gene machine
+ can design exactly fragment desired with sticky ends, labels and preferred codons - do not need an organism
- need to know the sequence of amino acids or bases
Steps of “in vitro” amplification = PCR
1- DNA denatured + hydrogen bonds broken = 94• temp
2- cooled to 50• + primers added to stop DNA rejoining + to initiate base pairing of new DNA nucleotides
3- heated to 74• + taq DNA polymerase (from a thermophile bacteria) forms new strand of DNA
4- cycle repeated
Why is taq DNA polymerase used
Able to withstand temps of up to 80• from a thermophile bacteria
Steps of “in vivo” amplification = vectors
1- vector DNA and DNA to be inserted are cut with the same restriction endonuclease = 2 strands are complimentary
2- DNA is annealed(cooled) with DNA Ligase = strands bind together
3- Vector DNA inserted into bacterial cell = transformer cell
4- promoter + terminator regions added to DNA to be cloned
- promoter regions stimulate RNA polymerase to transcribe this gene = producing desired protein
- terminator regions show where the DNA polymerase can stop transcribing = end of desired gene
What is added to genetically modified cells to show whether in vivo cloning/amplification has worked
Marker genes
Can code for fluorescence or antibiotic resistance
What vectors are usually used in “in vivo” cloning/amplification
Bacterial plasmids
Once fragment is in vector THEN it is placed into host eg: bacterial cell
So it can be transcribed
The vector and Host are different things
3 concerns of recombinant DNA
Agriculture = super weeds may evolve
Industry = may force small companies out of business through genetically modified crops + ppl may not get a say in whether they eat GM food
Medicine = designer babies
DNA probe
Single stranded DNA complimentary to a specific base sequence
What are DNA probes used for and how do they do this
Used to detect genes or harmful genes
Probe is complimentary to harmful gene = binds to gene in proscess known as DNA hybridisation
Probe is either fluorescently or radioactively marked
What do genetic counsellors do
Give advice to people on genetics
- give advice to parents concerned whether they will pass on faulty alleles
- advise women identified with carrying breast cancer gene
What are VNTRs
Variable number tandem repeats
Repeated base sequences that do not code for a protein
Why can VNTRs be used in genetic fingerprinting
Everyone has VNTRs but the number of repeats is unique for a person
Chance that 2 people have the same number is incredibly small
What is hybridisation and annealing
Hybridisation = 2 complimentary base sequences binding
Annealing = to cool /cooling such as in PCR
Steps of genetic fingerprinting (simple)
- Extraction = extract DNA from cell + amplify via PCR due to amount of DNA being small
- Digestion = restriction endonucleases cut DNA into fragments
- Separation = fragments are separated by size with gel Electrophoresis
- Hybridisation = radioactive or fluorescent probes added + bind to specific Fragments
- Development = depends on type of probes
Radioactive = X-ray film put over nylon membrane = radiation makes series of bars where fragments are
Fluorescent = visually can see bars
Stages of gel electrophoresis
- DNA extracted from individual + amplified
- DNA is cut into fragments + Flourescently labelled
- Fragrant inserted into wells in gel + covered in electrically conducting buffer
- DNA is negatively charged = when current applied DNA moves towards positive electrode
- Ladder forms and current stopped after certain amount of time = smaller fragments move further
6.exposed to UV light to show fragments
- the lengths of DNA fragments determined by number of repeats in a VNTR = this is unique per person = DNA fragments in an individual will move different distances = ladders are unique per person
The ladder = genetic fingerprint
Gene therapy
Genetic engineering technique = involves introducing new gene that translates for a desired protein that counteracts effect of disease cause by mutation
The gene introduced depends on whether mutated gene is dominant or recessive
Is PCR a form of recombinant DNA technology
No = does not involve transfer of DNA between organisms
PCR is a technique used to amplify DNA sequences outside of living organisms by heating + cooling
Somatic vs germline gene therapy
Somatic = alters body cells
Germline = alters sex cells (illegal)
Both involve vectors (plasmids) to insert target gene into genome of host/patient
Following the action of reverse transcriptase is cDNA single or double stranded
Reverse transcriptase used the mRNA template to produce SINGLE stranded cDNA = next DNA polymerase converts single stranded to DOUBLE stranded DNA
At what stage are promoter and terminator regions added in “vivo” cloning/amplification
Added to fragments of DNA before inserting them into host cells
Prompter regions have recognition sites for RNA polymerase to stimulate protein synthesis of specific protein = terminator genes are where RNA polymerase dissociates from DNA + ends transcription
