Genome Projects + DNA Technology Flashcards by Noah Repila Smith

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

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What is the genome

Complete set of genes present in an organism

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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

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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

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What is gene therapy

Used to replace defective genes with normal,healthy genes

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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

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Why have we sequenced the genome of other organisms such as zebra fish

To identify useful genes = may help us with our own problems

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Recombinant DNA technology

Transfer of DNA fragments from one organism to another

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Why can transferred fragments in Recombinat DNA technology be translated within recipient organism

DNA is universal

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Name 3 ways fragments can be produced

Reverse transcriptase
Restriction endonuclease
Gene machine

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What happened after fragments are produced

They need to be amplified
Either by :
1- in Vitro
2- in Vivo

(Vinland saga)

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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

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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

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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

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Producing fragments - Gene machine

DNA sequence entered into a computer = computer synthesis DNA strand

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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)

1. Extraction = extract DNA from cell + amplify via PCR due to amount of DNA being small 2. Digestion = restriction endonucleases cut DNA into fragments 3. Separation = fragments are separated by size with gel Electrophoresis 4. Hybridisation = radioactive or fluorescent probes added + bind to specific Fragments 5. 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

1. DNA extracted from individual + amplified 2. DNA is cut into fragments + Flourescently labelled 3. Fragrant inserted into wells in gel + covered in electrically conducting buffer 4. DNA is negatively charged = when current applied DNA moves towards positive electrode 5. 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