Gene technologies

Question 1

Define proteome

Answer 1

All the proteins produced in a given type of cell or organism at a given time

Question 2

Define recombinant DNA

Answer 2

DNA from two different sources joined together

Question 3

Define in vivo

Answer 3

Experiments performed within the living organism

Question 4

Define in vitro

Answer 4

Experiments performed test tubes/ petri dishes outside of the living organism

Question 5

Describe how using restriction enzymes to cut a fragment containing desired gene from DNA ( Step 1 - Isolation)

Answer 5

. Restriction endonuclease are enzymes which cut DNA at specific bas sequences
. Different restriction endonuclease cut DNA at different specific base sequences because the shape of the recognition site is complementary to the enzymes active site
. These recognition sites are palindromic
. If recognition site for these enzymes are either side of the DNA fragment you want, you can use restriction endonuclease to cut it out

Question 6

Define sticky ends

Answer 6

Short, single standard section at then end of a DNA molecule, the result of staggered cutting with a restriction endonuclease. Sticky ends can be used to bind DNA fragments together, if another DNA molecule has complementary sticky ends

Question 7

Define restriction endonuclease

Answer 7

Cut DNA at specific base sequences/ recognition sites. Breaks phosphodiester bonds. Produces sticky ends

Question 8

Define DNA ligase

Answer 8

Ligase joins DNA and plasmids/ vectors. Joins stick ends together. Reforms phosphodiester bonds

Question 9

Define recognition sites

Answer 9

. 4 to 8 base pairs long nucleotide sequence. Where restriction enzyme attaches and cuts

Question 10

Define palindrome

Answer 10

Base pairs reads the same in opposite directions

Question 11

Describe the conversion of mRNA to cDNA, using reverse transcriptase ( step 1 - isolation)

Answer 11

. Beta cells form islets of Langerhans in the human pancrease, where the insulin gene is transcribed into mRNA
. mRNA coding for insulin is extracted from the cell
. mRNA coding for insulin
. mRNA acts as a template on which cDNA strand is formed using reverse transcripts
. Single stranded cDNA
. New DNA nucleotides form hydrogen bonds, with complementary base pairs. Adenine to Thymine and Cytosine to Guanine
. DNA polymerase joins the new adjacent nucleotides / forms phosphodiester bonds and double stranded cDNA Is formed
. Copy of human insulin gene can now be inserted into host cells/ cloned

Question 12

What are the advantages of using reverse transcriptase

Answer 12

. mRNA is easy to obtain as it can be found outside of the nucleus
. Using mRNA isolated from Cytoplasm means intros have been removed

Question 13

Describe creating the gene in a gene machine ( step 1- isolation)

Answer 13

. Protein sequence analysis
. Amino acid sequence worked out
. mRNA sequence worked out
. DNA nucleotide base sequence worked out
. DNA nucleotide base sequence is uploaded into the computer
. Computer produces DNA base sequence by producing overlapping single strands of nucleotides called Oligonucleotides which can then be assembled into the gene

Question 14

What are the advantages of using the gene machine

Answer 14

. Faster to use the gene machine rather the using reverse transcriptase or restriction endonuclease
. There are more steps involved in isolation the gene/ mRNA as well as the enzyme catalysed reactions
. Highly accurate

Question 15

Define vector

Answer 15

A DNA carrier used to transfer DNA into host cells / organisms

Question 16

Define promoter region

Answer 16

Binding site for RNA polymerase on DNA

Question 17

Define RNA polymerase

Answer 17

Enzyme that joins RNA nucleotides to form mRNA during transcription

Question 18

Define transcription factors

Answer 18

Protein that binds to promoter region and RNA polymerase to begin transcription

Question 19

Define terminator region

Answer 19

Region of DNA that releases RNA polymerase to stop transcription

Question 20

Describe inserting the gene of interest into a vector ( step 2 )

Answer 20

. The most common type of vector is a plasmid which can be inserted into bacteria
. To prepare the vector for the gene of intreset the vector has to be opened by using the same restriction enzyme
. So the sticky ends are complementary
. Dna ligase is then used to recombine the 2 pieces if DNA
. This is known as recombinant DNA

Question 21

Describe chemical transformation ( part 1 of step 3)

Answer 21

Positive charge attracted to negative charge so DNA will not be repelled by membrane
. DNA has a negative charge
. Ca 2+ has a positive charge

Question 22

Describe heat shock ( part 2 of step 3 - transformation)

Answer 22

. Drop temperature- less kinetic energy/ less vibrations
. Quickly increase temperature- burst of kinetic energy/ lots of vibrations
. Creates gaps in membrane for DNA to move into the bacteria cell

Question 23

Describe step 4 : Identification of the transformed bacteria

Answer 23

. Some of the plasmids will have resealed without becoming recombinant- we don’t want this
. Some of the gene of interest will have resealed without becoming recombinant- we don’t want this
. There will be some recombinant DNA - we want this
. Some of the bacteria will not have transformed at all- we don’t want this`

Question 24

Define antibiotic resistance

Answer 24

The ability for microorganisms to withstand the adverse affect of antibiotics

Question 25

Define gene marker

Answer 25

Is a gene or DNA sequence with a known location on a chromosome ( plasmids) that can be used for identification. Can be modified to be fluorescent or radioactive

Question 26

Describe the procedure for PCR ( Polymerase chain reaction)

Answer 26

1) Separation of DNA strand- The mixture is heated to 95c which breaks hydrogen bonds between the bases, therefore separates the strands and produces single stranded DNA molecules = denatured 2) Addition of the primers- The temperature is reduced to 55-60c to allow primers to bind to the ends of the single stranded DNA molecules= Annealing 3) Synthesis of DNA- The temperature is increased to 72c. This enables the free DNA nucleotides to attach by complementary base pairing. The new DNA strands are built using the enzyme Taq polymerase which joins the phosphodiester bonds= extension. The first cycle is complete 4) The cycle is repeated 5) The 2 resulting DNA molecules make up the template DNA for the next cycle, thus amplifying the amount of DNA duplicated for each new cycle

Question 27

What are the pros and cons using in vitro (PCR)

Answer 27

Pros - Doesn't require a vector - Doesn't require living cells/ Bacteria - Very fast : hours to produce large quantities Cons - Must know sequence for primers to bind- must be studied before - Less accurate, any error will be copied in subsequent cycles - Can only accurately copy shorter sequences of bases- about 1000 base pairs

Question 28

Pros and cons using in vivo ( recombinant DNA )

Answer 28

Pros - More accurate, Mutations are very rare so less errors - Can copy unknown sequences that have not been studied before - Can accurately copy longer sequences of bases, about 2 million base pairs Cons - Require a vector, viruses may have harmful side effects - Require living cells/ bacteria - Slower, days or weeks to produce large quantities

Question 29

Uses of PCR

Answer 29

. Forensic analysis- DNA available from crime scenes . Screening- DNA from embryo can be amplified for genetic testing . Tissue typing . Tissue analysis from endangered/ extinct animal samples . Paternity testing . Identification of human remains

Question 30

Describe Gel electrophoresis

Answer 30

. DNA is hydrolysed by restriction endonuclease . Hydrolysed DNA placed in well at one end of an agarose gel . Gel is placed in a buffer . Electrical current is passed through the gel . DNA is negatively charged ( due to phosphate group) . DNA fragments migrate towards the positively charged end of the gel . Smaller DNA fragments migrate through the gel more quickly/ further than larger fragments

Question 31

Describe the visualising the DNA fragments: Southern Blotting and autoradiography

Answer 31

. Immerse gel in alkaline solution so two strands of DNA separated/ make DNA single stranded . Southern blotting- cover with nylon/ absorbent paper ( to absorb DNA) . DNA fixed to nylon/ membrane using UV light . Add radioactive or fluorescent probe to single stranded DNA . Show up using exposed to X-rays or UV light

Question 32

Define DNA hybridisation

Answer 32

. Two different strands of DNA/ RNA anneal/ join / bind together . e.g. DNA + Probe = hybridisation . Used to identify complementary alleles or how similar species are

Question 33

Define DNA probe

Answer 33

. Short single stranded of DNA ( 8-12 base pairs long) . Bases complementary with DNA/ allele/ VNTR

Question 34

Describe how are DNA probes used to identify alleles or genes

Answer 34

1. DNA extracted from sample 2. Amplified DNA using PCR 3. DNA hydrolysed into fragments using restriction endonuclease 4. Must leave alleles that is being screened for intact 5. DNA fragments separated by size using gel electrophoresis 6. Mixture put into wells on gel and electric current passed through 7. Immerse gel in alkaline solution so two strands of DNA separated as hydrogen bonds break 8. Southern blotting- cover with nylon/ absorbent paper to absorb DNA 9. DNA fixed to nylon membrane using UV light 10. Radioactive/ florescent marker/ probe added which is complementary to allele 11. Excess probes are washed away 12. Radioactive probe- identified using x-ray film/ autoradiography or florescent probe- identified using UV light

Question 35

Describe how to produce genetic fingerprints ( VNTR)

Answer 35

1. DNA extracted from sample 2. Amplify DNA using PCR 3. DNA hydrolysed into fragments using restriction endonuclease 4. Must leave VNTR that is being screened for intact 5. DNA fragments separated by size using gel electrophoresis 6. Mixture put into wells on gel and electric current passed through . Immerse gel in alkaline solution so two strands of DNA separated as hydrogen bonds break 8. Southern blotting- cover with nylon/ absorbent paper to absorb DNA 9. DNA fixed to nylon membrane using UV light 10. Radioactive/ florescent marker/ probe added which is complementary to VNTR 11. Excess probes are washed away 12. Radioactive probe- identified using x-ray film/ autoradiography or florescent probe- identified using UV light

Question 36

Uses of genetic fingerprinting

Answer 36

. Forensic science . Determine genetic relationships . Medical diagnosis . Determining genetic variability in a population . Animal and plant breeding

Question 37

Genetic fingerprinting and VNTR'S

Answer 37

. VNTR ( variable number tandem repeats) are repetitive sequences of non-coding DNA . Can vary by: - Number of bases in the repeated sequence varies - Number of times the sequence is repeated . Difference in VNTR can occur due to mutation and are retained as in the non-coding DNA . Restriction endonuclease cut at recognition sites on either side of the VNTR . Distance between recognition sites vary and are unique to each person . Produces different sized fragments

Question 38

What are the benefits with recombinant DNA technology in agriculture with example

Answer 38

. Higher yield . More nutritious crops . Reduces famine and malnutrition . Drought resistant crops . Golden rice , gene from soil bacteria and maize, high in beta-carotene makes vitamin A in humans. Vitamin A deficiently leads to blindness

Question 39

What are the concerns with recombinant DNA technology in agriculture

Answer 39

.Mono-culture- all crops susceptible to the same diseases and reduced biodiversity . Weeds become resistant to herbicides if transformed crops interbreed with wild crops

Question 40

What are the benefits with recombinant DNA technology in Industry

Answer 40

. Enzymes made in large quantities, for less money by transformed bacteria . Chymosin used in cheese making

Question 41

What are the concerns with recombinant DNA technology in Industry

Answer 41

. Purification of proteins may introduce toxins into the food industry . Labelling may not be clear so people may eat food made from transformed organisms . Large biotech companies using transformed organisms out complete smaller companies not using them

Question 42

What are the benefits with recombinant DNA technology in medicine

Answer 42

. Drugs and vaccines made from transformed organisms quickly, cheaply and in large quantities . Instead of using pig insulin, human insulin made using cloned human gene

Question 43

What are the concern with recombinant DNA technology in medcine

Answer 43

. Some big tech companies own the patent on particular GM seeds, may charge higher prices . Could make designer babies . Debate over who owns human genetic material once it has been removed from the body . Non GM crops may be contaminated by GM crops

Question 44

Define gene therapy

Answer 44

The treatment of genetic diseases by giving patients healthy copies of defective genes. There are two main types of gene therapy: Somatic ( body cells) and Germline (Sex cells/ gametes)

Question 45

Describe somatic gene therapy

Answer 45

. Functional gene inserted into body cells . Vectors required . If successful- only target cells are recombinant . Functional gene not passed onto child

Question 46

Describe germline gene therapy

Answer 46

. Functional gene inserted into sperm/egg . Vector would be needed but currently illegal so not tested . If successful- all cells are recombinant . Functional gene will be passed down to offspring

Question 47

Effectiveness and risks of gene therapy

Answer 47

. Short term treatment . Treatment must be separated . Difficult to get allele into the DNA in a functional state . Long term effects unknown . Vectors/ viruses cause harmful side effects . Modified cells/ vectors cause immune response/ rejection