Recombinant DNA Technology

Question 1

What is recombinant dna?

Answer 1

The transfer of fragments of dna from one organism to another. Since genetic code is universal, the dna can be translated/expressed in the new organism

Question 2

What are the 3 ways of isolating genes

Answer 2

1. Using reverse transcriptase 2. Using restriction endonucleases (enzymes) 3. Using a ‘gene machine’

Question 3

How is complementary dna made using reverse transcriptase

Answer 3

1. Select a cell type that usually expresses the gene of interest. 2. The cell’s mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) can be made using reverse transcriptase.3. Single stranded cDNA is isolated after hydrolysing the mRNA with an enzyme. 4. DNA polymerase is used to make a copy of the cDNA by using the cDNA as a template, resulting in a double stranded DNA molecule containing the gene of interest.

Question 4

Why is producing complementary dna using reverse transcriptase useful

Answer 4

when transferring a eukaryotic gene into prokaryotic cell because the cDNA does not contain any introns and prokaryotic cells do not have the ability to remove the intron sequences.

Question 5

How are restriction endonucleases used to cut dna into fragments.

Answer 5

Restriction endonucleases are enzymes that cut double-stranded DNA at specific base sequences called recognition sites. Restriction endonucleases can produce staggered ‘sticky ends’ which are useful for isolating a gene of interest and inserting it into the DNA of another organism.
Their restriction sites are palindromic. Some are blunt end endonucleases which don’t have sticky ends

Question 6

What is needed for dna insertion

Answer 6

A promoter sequence must be present or added in, where RNA polymerase and transcription factors bind, so that the gene can be expressed. A terminator sequence must be present or added in, where the RNA polymerase is released from the DNA at the end of the gene.

Question 7

What is the role of restriction endonucleases in formation of plasmids with donor dna/ the importance of sticky ends

Answer 7

They are used to cut the donor DNA to remove the gene of interest.They are used to cut the plasmid vector. Both the plasmid and donor DNA must be cut with the same restriction endonuclease that cuts are at the same base sequence recognition site/ both gene and plasmid have comp. sticky ends . This allows the sticky ends of both the plasmid and gene of interest to complementary base pair. (Ligase joins comp. sticky ends)

Question 8

What is the role of a vector

Answer 8

Vectors are used to transfer genes from one organism to another

Question 9

What is the role of dna ligase in production of plasmids containing donor dna

Answer 9

Ligase joins phosphodiester bonds to covalently bind the gene to the plasmid./ joins complementary sticky ends between gene and plasmid

Question 10

How are genes made using a gene machine

Answer 10

Base sequence of protein/ AA sequence determined and fed into computer (checked for bio safety/security to meet international/ethical standards. 2. Oligo nucleotides are made in small sections (small overlapping nucleotides) then hybridised to make the gene. Artificial ‘intron-free’ eukaryotic genes can also be made using a DNA synthesiser. Comp strand made using DNA polymerase, inserted into plasmid and checked for errors /sequenced

Question 11

What are the methods of isolating a gene that don’t contain introns and why is this significant for transferring dna into bacteria

Answer 11

Using reverse transcriptase and can create the genes using a gene machine. Using RE has introns as human dna has introns and important as Bactria can’t remove introns/splice pre mRNA so the protein cannot be expressed/functional

Question 12

Why is it faster to use gene machine than use reverse transcriptase

Answer 12

As there are a lot of extra steps to isolate the correct mRNA before being able to use reverse transcriptase

Question 13

What is transformation

Answer 13

When the bacterium contains the plasmid. Add dna from ligation/plasmid in with the bacteria, then heat shock.

Question 14

What are the possible outcome when mixing the bacteria with the plasmids

Answer 14

Only 1% will take up the plasmids when mixed together (99% non transformed). Very free are transformed and transgenic (contain the recombinant dna

Question 15

What does transgenic mean

Answer 15

When the bacteria contains the plasmid with the recombinant dna/ gene of interest

Question 16

Describe the nature of gene markers and how they work - for antibiotic resistance

Answer 16

Via insertional inactivation of a marker gene. Use a plasmid that has 2 different genes for antibiotic resistance. Use the same restriction endonuclease to cut the dna and plasmid in the middle of one of the antibiotic resistance genes. If the dna is transgenic- the resistance to one is inactivated but not for the other

Question 17

The different outcomes using antibiotic resistance marker genes if restriction site is in tetracycline resistance not ampicillin resistance

Answer 17

1. Non transformed: if the bacteria dies in both antibiotics 2.transformed but not transgenic: if the bacteria survives in both amp and tet 3. Transformed and transgenic: if bacteria dies in tet but survives in amp

Question 18

What is the process how you see which bacteria grow and which don’t

Answer 18

Replica plating: Dilute bacteria and plasmids then spread across agar plate and incubate to produce visible colonies. Imprint on a sterile material then imprint onto one plate with amp koi in and another with tetracycline then incubate and see which survives. Want colonies on the amp plate but not on tet

Question 19

The process of using fluorescent markers

Answer 19

Trainer green fluorescent protein from jellyfish into plasmid. Gene of interest inserted into GFP, any transgenic will not be fluorescent. No need for replica plating as cells don’t die , just view under uvlight or microscioe

Question 20

Describe the use of enzyme markers

Answer 20

Gene that produces the enzyme lactase is present which turns substrate blue. Gene transplanted into lactase gene. If plasmid is present- no lactase so no blue colour when gown on colourless substrate

Question 21

What do you do when you have identified transgenic bacteria

Answer 21

Growth/cloning of bacteria. Select transgenic and grow the, so they produce lots of the desired gene and protein

Question 22

What is one advantage of using fluorescent marker genes

Answer 22

You can quickly identify transformed bacteria using uv light

Question 23

In vitro and in vivi cloning and examples of each

Answer 23

In vivo describes a medical experiment or test that is preformed on a living organism (restriction es, rna transcriptase, markers etc.) in vitro is a medical experiment or study that is performed only in a lab dish or test tube (pcr..)

Question 24

Describe the polymerase chain reaction (pcr)

Answer 24

PCR) is a method of making millions of copies of a gene of interest in a test tube. used to amplify large quantities of a specific sequence of DNA from an initial minute sample. Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies

Question 25

How to work out the amount of dna each pcr cycle

Answer 25

2 to the power of n (number of cycles) if starting with 2 strands of dna. If starting with any other amount e.g. 10 do 10 x 2 to the n

Question 26

What is needed for the pcr to take place

Answer 26

1.A sample of DNA (containing the gene of interest) 2.DNA polymerase (Taq polymerase) - a heat stable enzyme from Thermus aquaticus, a bacteria that lives in hot springs. 3. Primers - short sequences of DNA to locate the gene of interest and get the DNA polymerase going. 4.Activated nucleotides - monomers to make the DNA. 5. A thermocycler - a machine that varies the temperature over a period of time.

Question 27

Describe the process of pcr

Answer 27

1.increasing the temp to about 95 ℃, denatures dna and breaks H bonds between c.b.p, separating strands. 2. Annealing/binding of the primers to dna at about 55 ℃ - complementary base pairing occurs at a specific sequence of the DNA sample (prevent the strands from rejoining. Allows DNA polymerase to attach/mark start and end of seq to be copied, start addition of nucleotides) 3.Synthesis of the DNA at 72 ℃ - optimum for Taq polymerase. Phosphodiester bonds formed and DNA extended in both directions until the temperature is raised and the cycle repeats.

Question 28

Describe the pattern of increase in dna numbers

Answer 28

There is a doubling of dna each cycle. Low numbers to start and then grows exponentially. Eventually levels off as nucleotides are used up so nothing to make comp chains

Question 29

why are two different primers required

Answer 29

As the base sequences at the ends of target sequences on dna are different (one at beginning one at end

Question 30

Why is the tag polymerase/dna polymerase good to be heat stable

Answer 30

So it doesn’t denature at high temperatures

Question 31

The advantages of in vitro and in vivo cloning

Answer 31

In vitro: Can be used on a minute sample of DNA, Extremely rapid (due to exponential increase), No complex culturing of bacteria - relatively simple to carry out. In vivo: Can be used to produce transgenic organisms, Accurate - fewer errors made than PCR, Low contamination - only the gene of interest is likely to be incorporated into the plasmid due to sticky ends, Can be used to produce useful products for medical use.

Question 32

Explain the technique of gel electrophoresis

Answer 32

lab technique used to separate DNA (or RNA) fragments based on mass / size. Samples placed in a block of gel and an electric current is applied which causes the samples to move through the gel. Smaller samples are less impeded by the gel matrix and move faster. DNA may be cut using REs – diff DNA samples will generate diff fragment lengths. Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide. Terminal in gel is positive

Question 33

What is a dna probe and how do they work

Answer 33

a short, single-stranded length of DNA that has bases complementary with dna/allele/gene. has some sort of label attached to it that makes it easily identifiable. Labels can be radioactive( detected using X ray film) or fluorescent tags. Probe binds to comp DNA sequence on target gene

Question 34

Explain how dna hybridisation is used to locate specific alleles of genes

Answer 34

Double stranded DNA is heated so it can be denatured and separated into single strands. On cooling, the complementary gene probes anneal & hydrogen bond to complementary sequences in the allele of interest.(Multiple copies of the probes can be made by PCR.) If the gene probe is complementary to a specific target sequence in the allele, it will hybridise and fluoresce or give off radioactivity if that sequence is present.

Question 35

Explain how labelled dna probes can be used to screen for heritable conditions or health risks

Answer 35

Gather dna sample, REs cut dna at recognition site/ spec base sequence , use electrophoresis to separate fragments, add dna probe with base sequence comp to harmful allele. Unbound probes are washed away. Bound probe c.b.p and use uv/radioactive film and if glows= got mutant allele

Question 36

What is the use of genetic screening in genetic counselling

Answer 36

Genetic counsellors help patients understand the results and implications of screening. Research family history of disease and advice likelihood of it in children. Medical tests/ ivf screening . Best course of cancer treatment personalised medicine

Question 37

What is genetic fingerprinting

Answer 37

In the non-coding regions of an individual’s genome there’s satellite DNA – long stretches of DNA made up of variable number tandem repeats (VNTRs). For everyone the number and length of VNTRs has a unique pattern. Probability two inds having same vntrs is very small - except twins. Sequence and loci don’t vary- two copies of each chromosome at each loci

Question 38

How is genetic fingerprinting carried out

Answer 38

1. Dna extracted 2. RE cut dna into fragments (blunt end) 3.fragments separated using gel electrophoresis 4. Fragments transferred from gel to nylon membrane 5. Hybridisation- dna probes added to label the fragments, bind to specific no. Repeat sequences 6. Membrane placed into X-ray film 7. Development of X-ray film reveals dark bands where radioactive probes attached

Question 39

The uses of genetic fingerprinting

Answer 39

Forensic science: DNA collected from the crime scene to determine which of the suspects was present -look for matches in the the banding patterns. The probability that someone else’s DNA might match that of the suspect can be calculated. presence of the DNA doesn’t mean that they committed the crime. Paternity testing: potential fathers compared with mother and child (child has some bands Same as mother, some same as dad- inherits half vntrs from each parent) plant and animal breeding: show diversity of pops and prevent undesirable inbreeding in farms/zoos (get most distantly related) and medical diagnosis e.g. huntindons as 38+ repeats means disease

Question 40

Two bands are usually seen for each person when looking at genetic fingerprinting , why would only one band be shown

Answer 40

As it is homozygous- same number of repeats for each allele and the same size

Question 41

Explain how recombinant dna is passed on

Answer 41

Dna is replicated with host cells dna if in nucleus, gene then passed to daughter cells by mitosis (dna of these cells are genetically identical p)

Question 42

How is a plant able to synthesise insect protein in recombinant dna

Answer 42

The genetic code is universal so insect dna can be transcribed and spliced by the plant cells. Mrna can be translated into a protein

Question 43

Describe how dna could be screened for all known harmful mutations

Answer 43

Use of PCR to amplify DNA sample/cut the dna using restriction enzymes. 3. Separate dna fragments using gel electrophoresis 4. Add labelled dna probe (fourecent marksr) which will hybridise to specific data sequence 5. Mutations idenfioed by fluorescence/radioactivity or positions of bands compared to known mutation dna