8.4 gene technologies

what is recombinant DNA technology

transfer of DNA fragments from one organism or species to another

explain why transferred DNA can be translated within cells of recipient organisms

genetic code is universal

transcription and translation mechanisms are universal

what is recombinant DNA technology

transfer of DNA fragments from one organism or species to another

explain why transferred DNA can be translated within cells of recipient organisms

genetic code is universal

transcription and translation mechanisms are universal

describe how DNA fragments can be produced using restriction enzymes

1. restriction enzyme cut DNA at specific base recognition sequences either side of desired gene

   - shape of recognition site complementary to active site

2. many cut in a staggered fashion forming sticky ends

describe how DNA fragments can be produced from mRNA

1. isolate mRNA from cell that readily synthesises the protein coded for by the desired gene

2. mix mRNA with DNA nucleotides and reverse transcriptase - reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA

3. DNA polymerase can form a second strand of DNA using cDNA as a template

suggest 2 advantages of obtaining genes from mRNA rather than directly from DNA removed from cells

much more mRNA in cells making the protein than DNA - easily extracted

in mRNA introns have been removed by splicing whereas DNA contains introns

• so can be transcribed and translated by prokaryotes who can’t remove introns by slicing

describe how fragments of DNA can be produced using a gene machine

synthesises fragments of DNA quickly & accurately from scratch without need for a DNA template

• amino acid sequence of protein determined, allowing base sequence to be established

these do not contain introns so can be transcribed & translated by prokaryotes

what does in vitro mean

outside a living organism

name an in vitro technique used to amplify DNA fragments

polymerase chain reaction

what does in vivo mean

inside a living organism

name an in vivo technique used to amplify DNA fragments

culturing transformed host cells

what is the reaction mixture used for PCR

DNA fragments

DNA polymerase

primers

DNA nucleotides

explain how DNA fragments can be amplified by PCR

mixture is heated to 95 C

• separates DNA strands

• breaking hydrogen bonds between bases

mixture is cooled to 55C

• allows primers to bind to DNA fragment template strand

• forming hydrogen bonds between complementary bases

mixture is heated to 72C

• nucleotides align next to complementary exposed bases

• DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

what happens to the amount of DNA in PCR after each cycle

DNA double

exponential increase - 2ⁿ

explain the role of primers in PCR

primers are short single stranded DNA fragments

complementary to DNA base sequence at edges of region to be copied

allowing DNA polymerase to bind to start synthesis

two different primers are required

suggest one reason why DNA replication eventually stops in PCR

there are a limited number of primers and nucleotides which are eventually all used up

what are the steps involved in amplifying DNA fragments in vivo

1. add promoter and terminator regions to DNA fragments

2. insert DNA fragments & marker genes into vectors (e.g. plasmids) using restriction enzyme and ligases

3. transform host cells by inserting these vectors

4. detect genetically modified / transformed cells by identifying those containing marker gene

5. culture these transformed cells allowing them to divide and form clones

why are promoter regions added to DNA fragments that are used to genetically modify organisms

allows transcription to start by allowing RNA polymerase to bind to DNA

• can be selected to ensure gene expression happens only in specific cell types

why are terminator regions added to DNA fragments that are used to genetically modify organisms

ensure transcription stops at the end of gene, by stopping RNA polymerase

what are the role of vectors in recombinant DNA technology

transfer DNA into host cells/ organisms

explain the role of enzymes in inserting DNA fragments into vectors

1. restriction endonucleases/enzymes cut vector DNA

   • same enzyme used to cut gene out so vector DNA & fragments have sticky ends that can join by complementary base pairing

2. DNA ligase joins DNA fragment to vector DNA

   • forming phosphodiester bonds between adjacent nucleotides

describe how host cells are transformed using vectors

plasmids enter cell

viruses inject their DNA into cells which is then integrated into host DNA

explain why marker genes are inserted into vectors

to allow detection of genetically modified/ transgenic cells

• if marker gene codes for antibiotic resistance, cells that survive antibiotic exposure are transformed

• if marker gene codes for fluorescent proteins, cells that fluoresce under UV light are transformed

as not all cells will take up vector and be transformed

how is recombinant DNA useful in medicine

GM bacteria produce human proteins - more ethically acceptable than using animal proteins and less likely to cause allergic reactions

GM animals produce pharmaceuticals - cheaper

gene therapy

how can recombinant DNA technology be useful in agriculture

GM crops resistant to herbicides - only weeds killed when sprayed with herbicide

Gm crops resistant to insect attack - reduce use of insecticide

GM crops with added nutritional value

Gm animals with increased growth hormone production

how is recombinant DNA technology useful in industry

GM bacteria produce enzymes used in industrial processes and food production

describe gene therapy

introduction of new DNA into cells, often containing healthy alleles

to overcome effect of faulty alleles in people with genetic disorder

suggest issues associated with gene therapy

effect is short lived as modified cells have a limited lifespan - requires regular treatment

immune response against genetically modified cells or viruses due to recognition of antigens

long term effect not know -side effects

• DNA may be inserted into other gene disrupting them - interfering with gene expression

why do humanitarians support recombinant DNA technology

GM crops increases yields - increased global food production - reduced risk of famine

gene therapy has potential to cure many genetic disorders

pharming makes medicines available to more people as medicines cheaper

why would environmentalists oppose recombinant DNA technology

recombinant DNA may be transferred to other plants

potential effects on food webs - reduces biodiversity

large biotech companies may control the technology and own patents

what are DNA probes

short, single stranded pieces of DNA

with a base sequence complementary to bases on part of target allele

usually labelled with a fluorescent or radioactive tag for identification

suggest why DNA probes are longer than just a few bases

a sequence of a few bases would occur at many places throughout the genome

longer sequences are only likely to occur in target allele

what is DNA hybridisation

binding of a single stranded DNA probe to a complementary single strand of DNA

forming hydrogen bonds

explain how genetic screening can be used to locate specific alleles of genes

1. extract DNA and amplify by PCR

2. cut DNA at specific base sequences using restriction enzymes

3. separate DNA fragments using gel electrophoresis

4. transfer to a nylon membrane and treat to form single strands with exposed bases

5. add labelled DNA probes which bind to target alleles

6. to show bound probe, expose membrane to UV light if a fluorescently labelled probe was used or use autoradiography if radioactive probe was used

what is gel electrophoresis

a method used to separate nucleic acid fragments or proteins

according to length/mass and charge

explain how gel electrophoresis can be used to separate DNA fragments

1. DNA samples loaded into wells with porous gel and covered in buffer solution

2. electrical current passed through - DNA is negatively charged so moves towards positive electrode

3. shorter DNA fragments travel faster so travel further

how can data showing results of gel electrophoresis be interpreted

run a standard with DNA fragments of known lengths under same conditions

compare positions of unknown DNA fragments to estimate size

shorter DNA fragments travel further

describe examples of the use of labelled DNA probes

screening patients for heritable conditions

screening patients for drug responses

screening patients for health risks

describe the role of a genetic counseller

1. explain results of genetic screening, including consequence of a disease

2. discuss treatments available

3. discuss lifestyle choice/ precautions that may reduce risk of genetic condition developing

4. explain probability of condition being passed onto offspring - enable patients to make informed decision about having children

what is personalised medicine

medicine tailored to an individuals DNA

increasing effectiveness of treatment

evaluate the screening of individuals for genetically determined conditions and drug responses

FOR:

• enables people to make lifestyle choices to reduce chances of diseases developing

• allow people to make informed decisions about having their own biological children

• allows use of personalised medicines, increasing effectiveness of treatment

AGAINST:

• screening for incurable diseases or diseases that develop later in life may lead to depression

• could lead to discrimination by insurance companies

• may lead to undue stress if patient does not develop disease

what are variable number tandem repeats (VNTR’s)

repeating sequences of nucleotides

found within non coding sections of DNA at many sites throughout an organism genome

why are VNTR’s useful in genetic fingerprinting

probability of two individuals having the same VNTR’s is very low

as an organisms genome contains many VNTR’s and lengths at each loci differ between individuals

explain how genetic fingerprinting can be used to analyse DNA fragments

1. extract DNA from sample and amplify by PCR

2. cut DNA at specific base sequences/ recognition sites using restriction enzymes

3. separate VNTR fragments according to length using gel electrophoresis

4. transfer to nylon membrane and treat to form single strands with exposed bases

5. add labelled DNA probes which bind with complementary VNTR’s

6. to show bound probe, expose membrane to UV light if a fluorescently labelled probe was used or use autoradiography if a radioactive probe was used

compare and contrast genetic fingerprinting with genetic screening

both use PCR to amplify DNA sample

both use gel electrophoresis to separate DNA fragments

both use labelled DNA probes to visualise specific DNA fragments

genetic fingerprinting analyses VNTR’s whereas genetic screening analyses specific alleles of a gene

explain how genetic fingerprinting can be used to determine genetic relationships

more closely related organism have more similar VNTR’s so more similarities in genetic fingerprints

paternity testing - father should share 50% of VNTR’s with child

explain how genetic fingerprinting can be used to determine genetic variability within a population

differences in VNTR’s arise from mutations, so more differences show greater diversity within a population

explain the use of genetic fingerprinting in forensic science

compare genetic fingerprint of suspects to genetic fingerprint of DNA at crime scene

if many bands match the suspect was likely present at the crime scene

explain the use of genetic fingerprinting in medical diagnosis

some VNTR patterns are associated with an increased risk of certain genetic disorders

explain the use of genetic fingerprinting in animal and plant breeding

show show closely related 2 individuals are so that inbreeding can be avoided

breed pairs with dissimilar genetic fingerprints