Gene Technology

Describe how functioning alleles can be inserted into a person's DNA to replace faulty alleles and transform their phenotype

- Extract the functional gene using restriction endonucleases - Replicate via PCR - Put in liposome or virus Either: - Introduce the functional DNA to the target cells - Introduce the functions gene into the gametes

Describe how a specific fragment of DNA can be produced by cutting DNA at specific recognition sequences using restriction endonucleases

- restriction endonucleases cut large DNA molecules into smaller fragemnts - each cuts DNA at its recognition sequence - different REs cut DNA at specific recognition sites because the shape of the sequence is complementary to the enzyme's active site - the recognition sequences are palindromic

Give the number of DNA fragments produced per PCR cycle, and use to calculate the number following 8 cycles

1 cycle = 2^1 = 2 2 cycles = 2^2 = 4 3 cycles = 2^3 = 8 4 cycles = 2^4 = 16 5 cycles = 2^5 = 32 6 cycles = 2^6 = 64 7 cycles = 2^7 = 128 8 cycles = 2^8 = 256

Describe the process of 'in vivo' gene cloning including the use of vectors (plasmids) restriction endonucleases, and DNA ligase

1. Extraction and purification of human DNA and of a bacterial plasmid (vector). 2. Cutting the DNA and vector with THE SAME restriction endonuclease to produce the same sticky ends. 3. Mixing the cut plasmid and human DNA, so that they join by complementary base-pairing to form a RECOMBINANT PLASMID. 4. Joining the sugar-phosphate backbone between the plasmid and the insert using the enzyme LIGASE. This is known as ligation. 5. Attachment of PROMOTER and TERMINATOR regions of DNA at the beginning and end of the gene. RNA polymerase attaches to DNA at a promoter region on DNA to enable transcription to occur. RNA polymerase stops transcribing at terminator sequences. 6. Putting the recombinant plasmid into a bacterium - known as 'transformation'. The recombinant plasmid solution and bacterial solution are mixed in calcium chloride. Some bacteria take up the plasmid so have been transformed. 7. Identifying the 'host' cells that have the gene, using marker genes. 8. Growing the bacteria which produces the insulin

Define the term gene probe

A short, single stranded DNA molecule with a complementary base sequence to a specific DNA fragment. It is labelled with a radioactive or fluorescent marker to enable its location.

Explain what is meant by term faulty allele

Alleles which have been mutated and will cause genetic disorder

Explain how the results of DNA fingerprints are used in accessing paternity, crime investigations, and determining the genetic variability within a population

Can compare blood tests with DNA of individuals. If the alleles match then individuals can be found.

What is a restriction endonuclease

Enzymes that cut DNA at a specific base sequence that it recognises. These are known as palindromic recognition sequences.

Explain how DNA fingerprinting can be used to select mates in conservation breeding programmes

Faulty alleles can be interpreted.

Outline why viruses and liposomes are suitable vectors for gene therapy but not plasmids

Liposomes containing corrected/correctly functioning gene fuses with the cell membrane, and releases the gene into the cell. Virus - if a foreign DNA fragment is introduced into the viral genetic material it will insert the foreign gene at the same time as its own genetic material, into the host's DNA. Viral DNA is cut using the same restriction endonuclease as those used to cut the fragment. The cut virus DNA and foreign gene are joined using DNA ligase. The virus then acts as a vector (DNA carrier).

Describe and explain the use of marker genes in the identification of transformed organisms

Marker gene - gene inserted into the vector which gives the transformed organism a specific characteristic that can be used to identify it.

Outline the process of replica plating when using two antibiotic resistance marker genes to identify recombinant organisms

Replica plating is the technique used to find the colonies of bacteria with the plasmid (ampicillin resistance) and with the inserted gene (sensitive to tetracycline). The ampicillin plate with colonies is gently pressed onto velvet. Some cells are transferred from each of the colonies onto the velvet. A tetracycline plate is then pressed on the velvet to pick up some cells. This is repeated with an ampicillin plate. All colonies will grow on the ampicillin as they have the plasmid. The colonies that don't grow on the tetracycline plate have the insulin gene / DNA fragment in. The colonies in those positions on the ampicillin plate are picked and used for growth in a fermenter. Another marker gene is a fluorescence gene. It codes for a fluorescent protein. Transformed organisms will fluoresce when a UV light is shone on them.

Describe how DNA probes are produced

Restriction mapping is the mapping of a piece of DNA showing recognition sites of specific restriction endonucleases and the numbers of bases between these sites. • Restriction enzymes are used to cut the DNA into fragments. • The fragments are separated by electrophoresis. • The pattern of the fragments allows the distance between the recognition sites to be determined. • DNA fragments are then sequenced separately (see below); the restriction map information is then used to piece fragments back together - giving the whole base sequence of a gene or region of DNA

Describe how DNA fragments can be separated according to size using gel electrophoresis

Smaller DNA fragments migrate through the gel faster than larger fragments. So they move further in the same time period. This process separates fragments according to their size across the gel.

Compare somatic and germ cell gene therapy

Somatic gene therapy (DNA transfer to our normal body tissue) and germ line gene therapy (DNA transfer to cells that produce eggs or sperm). The distinction is that the results of any somatic gene therapy are restricted to the actual patient and are not passed on to his or her children.

Define the term sticky ends

Some RE's cut double stranded DNA to give single stranded overhangs which are complementary.

Define the term palindromic

The base sequence on one strand will be the same as the sequence on the other strand but reading from the opposite direction

Define the term 'in vitro' gene cloning

The increase in number of a specific DNA fragment outside a living cell (PCR technique).

Define VNTR

Variable number tandem repeats - the regions of DNA between genes

Describe how specific DNA fragments can be identified using gene probes and hybridisation

• After electrophoresis, the bands need to be transferred to a nylon membrane as the agarose gel is very fragile. • Once on the nylon membrane, the double stranded DNA fragments are treated to break hydrogen bond between complementary base pairs, making them single stranded (their bases are exposed). • Gene probes with complementary base sequences to target DNA fragment are mixed with the nylon membrane (in solution). • The probes will bind to any complementary DNA fragment. This process is called DNA hybridisation. • Excess probes are washed away.

Explain the advantages of this reverse transcriptase

• Cells contain a maximum of two copies of each gene. If they are expressing the gene they will contain many mRNA molecules, with a complementary base sequence to the gene, so more cDNA can be obtained. • The mRNA is isolated from the cytoplasm of cells expressing the gene. It contains no introns (whereas the gene does). DNA fragments produced using mRNA as a template codes for a functioning protein without needing further modification. • As cells expressing a gene will have many copies of mRNA in their cytoplasm, you do not need to know the base sequence of the gene to copy it using this method.

Outline the process of producing a DNA fingerprint

• Extract DNA • PCR is used to amplify the VNTR regions within the sample. OR • Restriction endonucleases are used to cut DNA into fragments. Must produce blunt ends and cut outside of VNTR regions. • Separate DNA fragments according to size using gel electrophoresis. • Make fragments single stranded and add gene probes for VNTR's to allow positions of these fragments to be visualised as bands.

Describe how a specific fragment of DNA can be produced using complementary mRNA to produce cDNA using reverse transcriptase

• First mRNA is mixed with free DNA nucleotides reverse transcriptase. • Free DNA nucleotides bind to single stranded mRNA via complementary base pairing. • Reverse transcriptase joins DNA nucleotides together to form a newly synthesis DNA strand with complementary base sequence to the mRNA template. This complementary DNA is called cDNA. • The addition of further DNA nucleotides and DNA polymerase is then used to make cDNA double stranded.

Describe how a specific fragment of DNA can be produced by the polymerase chain reaction (PCR)

• Heat to 94 degrees C to break hydrogen bonds between two strands of DNA. (1) • Cool to 50 to 60 degrees C so that primers can bind to strands. (2) • Heat to 74 degrees C - the optimum temperature for the Taq polymerase. (3) • DNA polymerase joins DNA nucleotides together forming newly synthesised complementary strand for required section of DNA. • Two new copies of the DNA fragment are formed. This is one cycle. • The cycle starts gain, by heating to 95 degrees C, and this time four strands separate. • This is repeated many times. The number of copies of DNA fragments doubles with each cycle.

Describe how restriction mapping can be used to give the distance between restriction sites

• Restriction enzymes are used to cut the DNA into fragments. • The fragments are separated by electrophoresis. • The pattern of the fragments allows the distance between the recognition sites to be determined. • DNA fragments are then sequenced separately (see below); the restriction map information is then used to piece fragments back together - giving the whole base sequence of a gene or region of DNA.

Outline how individuals with faulty alleles can be identified using RE's, gel electrophoresis, labelled DNA probes and DNA hybridisation

• Restriction enzymes are used to cut the DNA into fragments. • The fragments are separated by electrophoresis. • The pattern of the fragments allows the distance between the recognition sites to be determined. • DNA fragments are then sequenced separately (see below); the restriction map information is then used to piece fragments back together - giving the whole base sequence of a gene or region of DNA.

Outline who, when, and why genetic screening could be used.

• Screen parents to determine if both are carriers of defective genes. • Screen embryos for faulty alleles. • Screen people in high risk families for oncogenes.

Outline key concerns of genetic engineering (including using antibiotic resistance marker genes, risk of creating toxic products within GE food sources, and risk of creating herbicides resistance weeds).

• Use of antibiotic resistance marker genes in GE could lead to the transfer of these genes to pathogens. This would reduce the range of effective antibiotics available in the treatment of diseases. • Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within GE food sources. • Introducing herbicide-resistance genes to crop plants could result in transfer to wild species when they interbreed, producing weeds resistant to herbicides ('super weeds').

Give the current limitations and the key concerns of gene therapy

• not all cells take up new allele, • not all cells express inserted allele, • only some tissue types are accessible i.e. the lungs. Treatment needs to be repeated - as cells die, they are replaced by cells with the faulty allele, • multiple treatments may be needed, • body can produce an immune response to the vector.