Gene Technology Topic 22

What stages are required to produce large quantities of pure proteins? Transferring DNA from one organism into another

- Isolation / production - of DNA fragments that contain the desired gene

- Insertion - of the DNA fragment into a vector

- Transformation - transfer of the DNA into suitable host cells

- Identification - of the host cells that have taken up the gene

- Growth / cloning - of the population of host cells

What makes obtaining DNA fragments difficult?

Must find the gene and then isolate it from millions of bases making up the genome

What are the method of producing DNA fragments?

- Using restriction endonucleases to cut the DNA fragments

- Conversion of mRNA into cDNA by reverse transcriptase

- Creating a gene in a gene machine based on known structures

Where are restriction endonucleases found?

- In bacteria to be used as defensive mechanisms

- They destroy the genetic material of invading viruses, inhibiting their ability to replicate

How do restriction endonucleases work?

- They cut DNA at a specific base sequence, called a recognition sequence (restriction site), breaking the phosphodiester bonds in the sugar phosphate backbone

- This can leave sticky ends

Define sticky ends

Single stranded DNA section that overhang at the end of a double stranded molecule

They are able to bind to another complementary sticky end

Where is reverse transcriptase found?

- Retroviruses have RNA but not DNA

- When they invade cells they must convert the RNA into DNA and they use reverse transcriptase

What does reverse transcriptase do?

Forms complementary cDNA strand from mRNA strand

What are the advantages of using reverse transcriptase to produce a gene rather than directly from the DNA using restriction endonucleases?

- mRNA at high conc so easier to identify and isolate compared to only 2 copies of the gene

- Only a few different types of mRNA in a cell but thousands of genes in DNA

- cDNA produced will have no introns so can be expressed in prokaryotes compared to the introns in DNA

Describe how double stranded cDNA is made from mRNA

- Reverse transcriptase and DNA nucleotides are added to the mRNA

- Complementary cDNA strand is made

- Treated with alkali to remove/digest mRNA/ break H bonds OR use enzyme to remove mRNA

- DNA polymerase makes complementary DNA strand to produce double stranded DNA

What is the function of DNA polymerase in the use of reverse transcriptase?

- Forms a second strand of DNA using single stranded cDNA as a template to form double stranded DNA

- Forms the phosphodiester bonds that create the sugar phosphate backbone to join the nucleotides

If you are trying to obtain the gene for insulin (mRNA for reverse transcriptase), why must pancreatic cells be used for this technique?

Only those cells contain insulin mRNA

Other cells don't form the mRNA

How is a gene machine used to make a gene?

- Amino acid sequence of a protein is determined

- mRNA codons are looked up

- Complementary DNA triplets are worked out and the gene is produced

- Gene is checked for errors

Give advantages of using the gene machine

- Any nucleotide sequence can be produced in a short period of time with good accuracy

- Genes are free of introns and other non coding DNA so can be transcribed and translated by prokaryotes

Give the disadvantage of using the gene machine and reverse transcriptase

- No sticky ends are produced which are useful to incorporate into recipient DNA

- However single stranded sections can be added to the ends of the DNA to create sticky ends

Describe the process of In Vivo cloning

- Preparing DNA fragment for insertion

- Insertion of DNA fragment into a vector

- Transformation of DNA into suitable host cells using vectors

- Identification of the transformed cells that have taken up the gene by using MARKER GENES

- Growth / cloning of the population of transformed cells

For In Vivo cloning:

1) Describe the preparation of the DNA fragment for insertion

- Adding a promoter to the start and a terminator sequence to the end of the gene

- Transcription factors and RNA polymerase can bind to the promoter region to begin transcription

- Terminator region can end transcription

For In Vivo cloning:

2) Describe the Insertion of the DNA fragment into a vector

- Vectors carry DNA into a host cell

- Vectors make sure the DNA can be replicated and expressed

- Viruses or liposomes (phospholipid vesicles) can be used as vectors but plasmids are generally used

- Same restriction endonuclease used to cut the gene and plasmid so have complementary sticky ends

- Complementary sticky ends join and DNA ligase joins the sugar phosphate backbone

What is a plasmid?

Small, circular DNA loop that is separate from the main DNA loop

Why are plasmids useful in gene technology?

- Can transfer into bacteria easily

- May contain genes for antibiotic resistance that can be used as marker genes

- Contains a replication origin so can replicate

- Contains recognition sites which restriction endonuclease can use to cut open

(Same restriction enzyme used to cut the DNA fragment and the plasmid)

Summarise how a gene is incorporated into a plasmid using restriction and ligase enzymes

- Rest endo cuts the gene from donor DNA with sticky ends

- Same rest endo cuts the plasmid in the middle of a marker gene

- The gene and plasmid are mixed together and join due to the same sticky ends

- Fragments are joined by DNA ligase to form recombinant plasmid

- Other products are formed eg some plasmids rejoin themselves + some fragments will form circles

Why is the plasmid cut in a marker gene? Eg antibiotic resistance gene

The antibiotic resistance gene is no longer expressed so doesn't have that characteristic

- Gene isn't expressed as gene is disrupted

For In Vivo cloning:

3) Describe Transformation

- This is the insertion of the extraneous DNA into the cell

- When plasmids successfully enter the cell, the cell is transformed

- The main method is to mix the plasmids and bacterial cells with calcium ions and give a rapid temperature change, known as heat shock

Why are calcium ions used in transformation?

- They bind to the negatively charged phosphate of the phospholipid bilayer, preventing repulsion with DNA

- Heat shock allows the plasmid to enter the cell

In transformation, why do only a few number of cells become transformed with the recombinant plasmid?

- Some cells don't take up anything

- Some cells take up the original plasmid without inserted gene that reformed

- Some cells will take up DNA fragments that didn't get incorporated into plasmids upon addition of ligase

For In Vivo cloning:

4) Describe the Identification of transformed cells using genetic markers

- Only a few cells take up the desired plasmid

- We can use marker genes to identify which cells have taken up the desired plasmid

- Marker genes often have antibiotic resistance genes or can code for fluorescent proteins or an enzyme whos action can be identified

What is an antibiotic?

A chemical produced naturally by some bacteria and fungi to kill or inhibit the growth of bacterial cells

Identifying transformed cells

Plasmids have 2 antibiotic resistance genes, A and B

Hybrid plasmids have disrupted gene B

Identify the cells with hybrid plasmids from the starting 4 cell types

- Add antibiotic A

- This means only cells with regular plasmids and hybrid plasmids will survive

- Cells with no plasmids or circular DNA fragments die (untransformed cells)

- Use replica plating to get a copy of the agar plate

- Add antibiotic B

- Only cells with regular plasmids survive

- Compare the replica plate with the tested plate and where the colonies are missing is where the hybrid plasmids are present (in those cells)

- This is because the hybrid plasmid has disrupted gene B so isn't resistant to antibiotic B

- Nowadays marker genes that are fluorescent are attached so we can visible see where the cells that have taken up the hybrid plasmids are

For In Vivo cloning:

5) Describe growing bacteria on a large scale

- The copied cells will be clones, capable of producing the product of the inserted gene

What do we do to extracted DNA before using it further?

- Many copies of them need to be made

- The process is polymerase chain reaction, PCR

- This makes identical copies of DNA by in vitro cloning

Describe the process of In Vitro Cloning

- DNA fragment is added to a thermocycler with DNA polymerase, free DNA nucleotides and primers

- Temp starts at 95*C to break H bonds and separate the DNA strands

- Temp reduced to 40*C so primers can attach to complementary base sequences at the ends of the single strands

- DNA polymerase attaches and the temp is raised to 75*C which is optimum for the enzyme. Free DNA nucleotides attach to the single stranded templates by complementary base pairing and DNA polymerase makes the new strands by joining the free nucleotides together with phosphodiester bonds

- The cycle is repeated so the DNA is doubled each time

How do you calculate number of DNA copies from In Vitro cloning?

Number of DNA copies = 2^n

n is the number of cycles

For in vitro cloning, how do the DNA polymerase enzymes survive the high temperature?

They are obtained from a species that can tolerate high temperatures

What are primers?

From in vitro cloning

Short sequences of single stranded nucleotides, with a specific base sequence

The are complementary to the ends of the fragments to be copied

They allow DNA polymerase to attach and starts DNA synthesis

Give examples of how plants can be modified using recombinant DNA technology?

- Adding genes for resistance to herbicides, insect pests and viral disease in various plant species

- Adding genes to develop tolerance to extreme environmental conditions eg extreme temp or drought

- Adding genes to improve the nutrient content of foods

Give examples of direct use of genetically modified bacteria

- Increase quantity of antibiotics and the rate at which they are made

- To produce hormones such as insulin, growth hormone, oestrogen and testosterone. This avoids killing animals and has fewer side effects on the patient

- To produce many enzymes used in the food industry. eg amylase to break down starch for beer

Give examples of how animals are genetically modified

Desired gene is inserted into the fertilised egg so that all body cells will have a copy of the gene

- Introduce a growth hormone gene so animals grow larger and faster to improve food production

- Introduction of genes to code for human proteins in milk producing animals. This means the proteins can be produced and used for humans who may lack these proteins

Why are milk producing animals genetically modified to produce desired proteins?

- Protein will be secreted in the milk

- This can then be extracted

- Avoids using invasive procedure to kill the animal

Describe gene therapy

- Potential treatment for genetic diseases by altering the genotype

- Cause of disease is targeted rather than the symptoms

- Not replacing the defective gene but adding the functional gene to work alongside

- Both healthy and non functional protein will be produced so cells may be able to function correctly

Why does gene therapy only work for conditions caused by recessive alleles?

- Existing alleles still present

- If these alleles are dominant they would still be expressed to cause the symptoms even if a normal recessive allele is added

Give the arguments for using recombinant DNA technology

- Microbes, plants and animals can be modified to produce useful substances such as insulin

- GM crops can help prevent disease eg by producing rice with higher vitamin A conc

- Gene therapy can be used to treat genetic diseases eg cystic fibrosis

Give the arguments against using recombinant DNA technology

- Impossible to predict the risks when releasing GM organisms into the environment

- Genes mutate so useful ones may become harmful

- Is it immoral to tamper with genes? Where is the line?

What is gel electrophoresis used for?

To separate different pieces of DNA based on their length

- analyses DNA fragments to produce genetic fingerprint

- locate defective genes such as those for specific diseases - using gene probes

Describe the process of gel electrophoresis

- DNA samples are amplified using PCR then cut into fragments using restriction enzymes

- Fragments are placed into wells at one end of the slab of gel

- Electric current is passed through the gel - DNA is attracted to the positive electrode

- Molecules diffuse through the gel, smaller lengths move faster than larger lengths so smaller move further down the gel in a given time

In gel electrophoresis, why does the DNA move to the positive electrode?

Nucleotides contain negatively charged phosphate group

In gel electrophoresis, how is the DNA visible?

Separated fragments can't be seen so must be marked

- DNA stained with colourless or fluorescent chemical

- DNA samples can be radioactively labelled and X ray film can identify positions of the fragments. Shows dark bands on the autoradiograph

In gel electrophoresis, how can DNA length be found?

Use size markers in a separate well to compare lengths of unknown fractions to bands of known length

Describe genetic fingerprinting

- Used to compare the VNTR's between genes (non coding DNA)

- Most individuals have a different number of repeats so each VNTR is unique

- So they move a different distance in gel electrophoresis

- So VNTR's are used to compare the DNA in forensic science

Why do closely related individuals have VNTR's that are more similar than individuals who are not related?

- DNA inherited from same individuals eg parents

- Within the family they inherit some of the same chromosome so VNTR's

Name the 5 stages of genetic fingerprinting

1) Extraction of DNA

2) Digestion

3) Separation

4) Hybridisation

5) Development

For genetic fingerprinting:

1) Describe extraction of DNA

- Sample of DNA is is extracted from tissues eg blood

- PCR then increases the quantity of DNA

For genetic fingerprinting:

2) Describe Digestion

- DNA is cut into fragments using restriction endonucleases

- They are chosen to cut close to but not within VNTR sequences

For genetic fingerprinting:

3) Describe Separation

- Gel electrophoresis used to separate DNA fragments

- Double stranded DNA fragments separated into single strands by immersing the gel in alkali

For genetic fingerprinting:

4) Hybridisation

- Radioactive or fluorescent DNA probes are used to bind with the DNA

- Probes have complementary DNA sequences to the VNTR's

- Many different probes can be used, to bind with different VNTR sequences at different regions

- RECALL it is the number of VNTR repeats that varies rather than the VNTR sequence itself

For genetic fingerprinting:

5) Development

- X ray film placed over the sample

- Film exposed to radiation from the DNA probes attached to DNA samples

- Banding patterns arise

- If fluorescent probes are used the sample is exposed to UV light

- DNA from crime scenes can be compared to suspect DNA to see if banding patterns are identical

For genetic fingerprinting, how can this be used in forensic science?

- Analyse DNA at crime scenes

- Find out who was present at the scene (doesn't show whos guilty of crime)

- Some DNA may belong to innocent bystanders who were at the scene

For genetic fingerprinting, how can this be used in determining paternity/ genetic relationships?

- Individuals inherit half the DNA from mother and half from father

- Each band on the genetic fingerprint corresponds to the band on a parents genetic fingerprint

- Closely related will have similar genetic fingerprint

For genetic fingerprinting, how can this be used in medical diagnosis?

- Genetic fingerprints can help diagnose diseases that match fingerprints of people with various forms of the disease

eg Huntington's disease which usually presents symptoms in middle age so can be identified early

- Can be used to determine the nature of a microbial infection by comparing the fingerprint of the microbe to know pathogens

For genetic fingerprinting, how can this be used in plant and animal breeding?

- Used to prevent inbreeding

- Can identify how closely related individual animals are at different zoos

- Can identify animals / plants with particular allele of desirable gene

Describe DNA probes

Single stranded, short section of DNA nucleotides with a complementary base sequence to the gene / allele being screened for

- DNA probe will bind to the target gene / allele if it is present in the sample of DNA

- To identify where the DNA probe has attached, the probe has a label which can be radioactive or fluorescent

Describe genetic screening

Involves using DNA probes that are produced by PCR to screen patients for clinically important genes / alleles

How is genetic screening different to genetic fingerprinting?

- Coding DNA testing in screening / VNTRs tested in fingerprinting

- Only one probe used to complementary base sequence in the mutant gene being tested for in screening. DNA fragments of interest can be removed from the gel and purified

On genetic screening, how can you tell if a gene is homozygous of heterozygous?

The homozygous band will be more intense in fluorescence or radioactivity

This is due to the 2 copies of the allele being present

Why may it be useful to screen for oncogenes?

- May have family history of cancer

- Can start having regular check ups for early diagnosis and treatment of cancer

- Can undergo pre emptive surgery

- Can be offered counselling

- Can alter lifestyle to reduce developing cancer risk

How can genetic screening be used in personalised medicine?

- Provides advice and healthcare based on the genotype of a person

- Can be used to decide which drugs are more effective and at what dosage