Biology - Chapter 21: Recombinant DNA Technology

What does recombinant DNA technology involve?

The transfer of fragments of DNA from one organism, or species, to another

What problems are associated with introducing extracted chemicals into patients to treat a deficiency? (3)

1. Rejection

2. Risk of infection

3. Cost

Recombinant DNA

The DNA produced after the DNA of two organisms has been combined (by having the genes of one organism isolated, cloned and transferred to another)

Transgenic organism

An organism that carries recombinant DNA - it is the recipient of the transferred DNA

Alternative name for a transgenic organism

Genetically modified organism

Why can DNA be transferred between organisms and function normally?

The genetic code, as well as translation and transcription mechanisms, are universal

Outline the process of making a protein using the DNA technology of gene transfer and cloning (5)

1. Isolation of DNA fragments that have the gene for the desired protein

2. Insertion of the DNA fragments into a vector

3. Transformation - DNA fragments are transferred into suitable host cells

4. Identification using gene markers to identify the host cells that have successfully taken up the gene

5. Growth/cloning of the population of host cells

What feature of the structure of different proteins enable them to be separated by gel electrophoresis? (3)

1. Number of amino acids

2. Charge

3. Different R groups

3 methods of producing DNA fragments

1. Conversion of mRNA to cDNA using reverse transcriptase

2. Cutting fragments containing the desired gene from DNA using restriction endonucleases

3. Creating the gene in a gene machine (usually based on a known protein structure)

How is reverse transcriptase used to produce DNA fragments? (3)

1. mRNA of the desired protein is extracted from the cell that produces the protein

2. Reverse transcriptase is used to produce cDNA from the RNA

3. DNA polymerase forms the complementary strand of DNA by catalysing the condensation reaction between the aligned DNA nucleotides

cDNA

Complimentary DNA

How are restriction endonucleases used to produce DNA fragments?

It cuts the DNA double strand at the recognition sequence, leaving either blunt ends or sticky ends

Restriction endonucleases

Enzymes that cut up DNA

Why are there many types of restriction endonucleases?

Each one cuts a DNA double strand at a specific sequence of bases - the recognition sequence

Recognition sequence

A specific sequence of bases where restriction endonucleases cut the DNA strand

How do restriction endonucleases cut the DNA to form blunt ends?

The cut occurs between two opposite base pairs, leaving two straight edges.

How do restriction endonucleases cut the DNA to form sticky ends?

The cut occurs in a staggered fashion, where each strand in the DNA has exposed, unpaired bases - the base sequences of the ends are opposites of each other i.e. palindromes

How is the gene machine used to produce DNA fragments? (9)

1. Desired seqquence of nucleotide bases is determined from the desired protein to be produced

2. mRNA codons are looked up, complementary DNA triplets are worked out

3. Desired nucleotide bases are fed into a computer

4. Sequence is checked for biosafety and biosecurity to ensure it meets international standards + ethical requirements

5. Computer designs oligonucleotides, which are assembled into the gene

6. Oligonucleotides are assembled into the required sequence by adding the nucleotide one at a time, producing a gene

7. The gene is replicated and the complementary DNA strand produced using the PCR

8. The gene is then inserted into a bacterial plasmid using its sticky ends

9. Genes are checked using standard sequencing techniques and those with errors are rejected

Oligonucleotides

Small, overlapping single strands of nucleotides

2 advantages of using the gene machine to produce DNA fragments

1. Any sequence of nucleotides can be produced in a very short time with great accuracy

2. The genes are free of introns and other non-coding DNA

In vivo

Transferring the fragments to a host cell using a vector

In vitro

Using the polymerase chain reaction

What happens if the same restriction endonuclease is used to cut DNA?

All the fragments produced will have ends complementary to one another, so the single-stranded end of any one fragment can be joined to the single-stranded end of any other fragment

How are the complementary bases of two sticky ends joined together?

DNA ligase is used to bind the phosphate-sugar backbone of the two sections of DNA and unite them as one

Why are sticky ends useful?

As long as the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism

What must happen for transcription to take place?

RNA polymerase must attach to the DNA near a gene

Promoter

A region of DNA that acts as the binding site for RNA polymerase as well as transcription factors

Terminator

A region of DNA that releases RNA polymerase and ends transcription

What must be attached to a DNA fragment so that it will transcribe mRNA and produce a protein?

A promoter and terminator

Vector

Carrying unit used to transport the DNA fragment into the host cell

Give an example of a vector

A plasmid

What restriction endonuclease is used to cut the plasmid?

The same one as the one used to cut the DNA fragment so the sticky ends will be complementary to each other

How are DNA fragments inserted into a vector? (2)

1. Restriction endonuclease used to cut plasmids to produce sticky ends - same one as the one used to cut DNA fragment

2. DNA ligase joins sticky ends of the DNA and plasmid together, forming recombinant DNA

How is DNA introduced into host cells? Why? (2+1)

1. Plasmids and bacterial cells are mixed with calcium ions

2. Temperature is also increased

This makes the bacterial membrane permeable so the plasmids can pass through into the cytoplasm

Transformation

The introduction of recombinant DNA into host cells

3 reasons why not all bacterial cells may possess the DNA fragments for the desired gene

1. Only a few bacterial cells will take up the plasmids when mixed together

2. Some plasmids close up without incorporating the DNA fragment

3. Some DNA fragments join together to form its own plasmid

How can you find out which bacterial cells have taken up plasmids? What is an issue associated with this? (3+1)

1. All bacterial cells are grown on a medium containing an antibiotic

2. Bacterial cells with plasmids have the gene for antibiotic resistance, so can break the antibiotic down and survive

3. The bacterial cells without the plasmids die

This includes bacterial cells that have taken up the plasmid and gene as well as cells that have taken up just the plasmid

3 examples of marker genes

1. Genes for antibiotic resistance

2. Genes that produce a fluorescent protein

3. Genes that produce an enzyme with an identifiable action

How is replica-plating used to identify cells with the recombinant DNA? (4)

1. In cells with the recombinant DNA, the gene for resistance to another antibiotic is cut due to the inserted DNA

2. Replica-plating is used to replicate the arrangement of the bacteria colonies

3. One of the plates is grown with a sample with that antibiotic

4. The colonies that die have recombinant DNA, so are isolated and used

How are fluorescent markers used to identify cells with the recombinant DNA? (3)

1. Gene that codes for a fluorescent protein is transferred to the plasmid

2. Gene to be cloned is inserted in the middle of the gene for the fluorescent protein, so cells with the recombinant DNA do NOT glow

3. The cells that do glow do NOT have the recombinant DNA, so are discounted

How is enzyme-markers used to identify cells with the recombinant DNA? (3)

1. Gene that codes for an enzyme is transferred to the plasmid

2. Gene to be cloned is inserted in the middle of the gene for the enzyme, so cells with the recombinant DNA do NOT change colour as a result of the enzyme’s action

3. The cells that do change colour do NOT have the recombinant DNA, so are discounted

Polymerase Chain Reaction

A method of copying fragments of DNA

Why is PCR rapid and efficient?

It is automated

3 things that need to be added to the DNA fragment in PCR

1. DNA polymerase

2. Primers

3. Nucleotides

Describe the process of PCR (4)

1. Mixture of DNA polymerase, DNA nucleotides and primers

2. Temperature is heated to 95°C to break the hydrogen bonds between the DNA strands, causing the strands to separate

3. Temperature is cooled to 55°C so the primers bind to the ends of the DNA strands

4. Temperature is heated to 72°C so DNA polymerase joins the adjacent nucleotides together by catalysing the condensation reaction between them

What happens to the number of DNA molecules after a cycle of DNA polymerase?

It doubles

2 advantages of in vitro cloning

1. It is extremely rapid

2. It does not require living cells - only need base sequence of DNA

4 advantages of in vivo cloning

1. Useful if want to introduce a gene into another organism

2. Involves almost no risk of contamination

3. It is very precise and only cuts out specific genes

4. Produces transformed bacteria that can be used to produce large amounts of gene products

2 ways to find out where a gene is located

By using labelled DNA probes or DNA hybridisation

2 most commonly used probes

1. Radioactively-labelled probes: use isotope ¹²P - probe identified with X-ray film exposed by radioactivity

2. Fluorescently-labelled probes: emit light under certain conditions

When does DNA hybridisation take place?

When a section of DNA/RNA is combined with a single stranded section of DNA which has complementary bases

What might DNA probes be used to screen for? (3)

Heritable conditions, drug responses or health risks

What are the benefits of genetic screening? (3)

People at a greater risk of developing a disease can make informed decisions about their lifestyle and future treatment

1. They can be checked more regularly, so may be diagnosed earlier and have a better chance of successful treatment

2. Personalised medicine: people can receive medical advice and treatment specific to their genotype

Genetic counselling

A form of social work where people are provided with advice and information to enable them to make personal decisions about themselves or their offspring

3 things genetic screening can detect with regards to cancer

1. Oncogene mutations: can determine type of cancer person has and so most effective drug / radiotherapy available

2. Gene changes that predict which patients are more likely to benefit from certain treatments and have the best chance of survival

3. A single cancer cell among normal cells, so can tell whether someone is at risk of relapsing

Why might you use the gene machine over using reverse transcriptase / restriction endonuclease?

It is faster to use the gene machine than all the enzyme-catalysed reactions

Why would you not use restriction endonucleases to obtain DNA fragments when the fragments are going to be inserted into bacterial plasmids? (2)

1. Human DNA contains introns

2. The bacteria cannot splice pre-mRNA

Why is it important that inserted genes are only expressed in specific places? (2)

1. So protein can be harvested - it may be easy to extract

2. Producing the protein in other cells / elsewhere, it may damage other cells, causing harm

When trying to carry out PCR for a sample of genetic material, why might you need to produce a variety of primers?

The DNA/RNA base sequences differ, so different complementary primers are needed

Why might bacteria not be able to produce every human protein? (3)

1. Do not have required transcriptional factors, so cannot carry out transcription and produce mRNA

2. Cannot splice pre-mRNA, so cannot remove introns

3. Do not have Golgi apparatus, so cannot modify proteins

Variable number tandem repeats

DNA bases which are non-coding

VNTRs

Variable Number Tandem Repeats

What is the probability of two individuals having identical VNTR sequences? Why?

Very low - for each individual, the number and length of VNTRs has a unique pattern. This is different for all individuals except identical twins.

How does relation affect the pattern of VNTR sequences?

The more closely-related two individuals are, the more similar the VNTRs will be

What is gel electrphoresis used for?

To separate DNA fragments according to their size

In gel electrophoresis, how does the size of the fragment affect the speed they move at? Why?

The larger the fragment, the slower they move - this is due to the resistance of the agar gel

What must be done before larger genes and whole genomes go through gel electrophoresis?

They must be cut into smaller fragments by restriction endonucleases

What is genetic fingerprinting used for? (4)

1. Determining genetic relationships and the genetic variability in a population

2. Forensic science

3. Medical diagnosis

4. Plant and animal breeding

Why is genetic fingerprinting used to determine genetic relationships and the genetic variability in a population? (3)

• The more closely related individuals are, the closer the resemblance of their genetic fingerprints

• Relatives will share some bands of their genetic fingerprints

• A population that has members with very similar genetic fingerprints have little genetic diversity

Why is genetic fingerprinting used in forensic science?

DNA found at the crime scene can be analysed and compared to identify someone at the scene of the crime

Why is genetic fingerprinting used in medical diagnosis? (2)

• Can be used to determine if someone has base sequence / alleles responsible for a genetic disorder, the probability of them developing and when

• Can be used to identify the nature of a microbial infection by comparing fingerprint of the microbe with other similar pathogens

Why is genetic fingerprinting used in plant and animal breeding? (2)

• Can be used to select individuals for breeding if they have a desirable allele / prevent individuals with undesirable alleles from breeding

• Can be used to determine the paternity of an animal, establishing its pedigree

Describe how genetic testing / genetic fingerprinting / DNA screening is carried out. (7)

1. DNA extracted from sample

2. PCR used to amplify DNA sample

3. DNA cut into segments using restriction endonucleases

4. DNA fragments separated using electrophoresis according to size / length

5. DNA strands separated by an alkali

6. Labelled DNA probes are applied and bind to the sample (by DNA hybridisation)

7. Mutations identified using X-ray film due to radioactivity OR bands compared to DNA sample (e.g. one with mutations)

DNA primers

Short lengths of single-stranded DNA

Why are DNA primers added in PCR? (3)

1. To mark the beginning and/or ends of the part of DNA needed

2. So enzymes / nucleotides can attach

3. To keep the DNA strands apart

What is special about the DNA polymerase used in PCR?

It is thermostable

Why is it important that the DNA polymerase enzyme used in PCR is thermostable?

It would not be denatured - in PCR, it must be heated to 95°C, so it must be able to withstand high temperatures

Why is PCR needed before carrying out genetic fingerprinting?

Only small sample of DNA is obtained / would need more DNA for analysis - PCR gives many copies

What must be done if a DNA sample is contaminated before obtaining a genetic fingerprint? Why?

Need to identify “required” DNA from rest as other DNA is present

What is an advantage of including promoter DNA when inserting recombinant DNA for a desired gene into an organism?

It ensures that the desired gene is only expressed in the needed organ and not elsewhere

How are DNA probes used to identify what cells contain a gene? (6)

1. Extract DNA

2. Add restriction endonucleases

3. Separate fragments using gel electrophoresis

4. Treat DNA using an alkali to separate the strands and form single strands

5. Probe anneals to gene

6. Use X-ray film to show bound probe

DNA probe

A short single strand of DNA with bases complementary to the base sequence of DNA

How do DNA probes work? (3 - brief answer)

1. Probes are single stranded

2. They have a specific base sequence complementary to the DNA

3. They bind to the DNA and can be identified

Why would it be useful to identify the nature of a disease using genetic fingerprinting? (3)

1. To see if pathogen is resistant to treatment so can prescribe an effective one (e.g. antibiotics)

2. To see if a vaccine would work and what vaccine to use so can reduce the spread

3. To test others and see if they have the same strain so the spread of the disease can be controlled + people can be vaccinated/treated

When inserting a plasmid with recombinant DNA into an organism, why should a marker gene be used?

Because not all cells will successfully take up the plasmid so can identify the ones that did

What are vectors used for in genetic engineering?

To carry genes from one organism to another