21 Recombinant DNA Technology

Question 1

Recombinant DNA

Answer 1

Techniques have been developed to isolate genes, clone them and transfer them into microorganism, which are grown to provide continuous production of desired protein.
DNA of different organisms combined in such a way is called recombinant DNA, resulting organism known as transgenic or genetically modified organism (GMO)

Question 2

How can DNA that has been transferred from one organism to another function normally

Answer 2

Genetic code same in all organisms; universal so can be used by all living organisms

Question 3

Can proteins be made normally when DNA transferred from one organism to another

Answer 3

Yes because this is also universal, mechanisms of transcription and translation are same in all living organisms.
Indirect evidence for evolution

Question 4

Use of reverse transcriptase to isolate genes

Answer 4

A cell that readily produces protein that is selected (e.g. B cells of islets of Langerhans).
These cells have large quantities of relevant mRNA which is therefore more easily extracted.
Reverse transcriptase used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because made up of nucleotides that are complementary to mRNA.
To make other strand of DNA, enzyme DNA polymerase used to build up complementary nucleotides on cDNA. This double strand of DNA is required gene.
SEE CARD 1

Question 5

Restriction endonucleases

Answer 5

Some bacteria defend themselves against infection from viruses by producing enzymes that cut up the viral DNA

Question 6

Blunt ends

Answer 6

There are many types of restriction endonucleases. Each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence, sometimes this cut occurs between two opposite base pairs, leaving two straight edges.
SEE CARD 2

Question 7

Sticky ends

Answer 7

Some Restriction endonucleases cut DNA in a staggered fashion leaving an uneven cut in which each strand of DNA has exposed unpaired bases
SEE CARD 2

Question 8

What is a palindrome

Answer 8

If you read both unpaired passe at each end from left to right the 2 sequences are opposites of one another. Recognition sequence is therefore palindromic sequence

Question 9

Action of restriction endonucleases

Answer 9

SEE PG 268

Question 10

How to manufacture genes in lab in gene machine

Answer 10

1. The desired sequence of nucleotide bases of gene is determined from protein we want to produce. Amino acid sequence determined, mRNA codons looked up and complementary DNA triplets worked out.
2. Sequence of bases fed into computer, sequence checked for biosafety and bio security.
3. Computer designs series of small overlapping single strands of nucleotides, called oligonucleotides, which are assembled into gene. One nucleotide is added at a time. Gene does not have introns or non coding DNA.
4. Gene is replicated using polymerase chain reaction, which constructs complementary strand of nucleotides to make double stranded gene. It then multiplied this many times to give many copies.
5. Using sticky ends gene can be inserted into bacterial plasmid, which acts as vector for gene, so it can be stored cloned or transferred to other organism.
6. Genes are checked and those with errors are rejected.
   SEE PG 270

Question 11

Advantages of gene machine are

Answer 11

Any sequence of nucleotides can be produced in very short time and with great accuracy.
These artificial genes are also free of introns and other non coding DNA so can be transcribed and translated by prokaryotic cells.

Question 12

Two ways to clone

Answer 12

In vivo; by transferring the fragments to a host cell using vector.
In vitro; using the polymerase chain reaction

Question 13

Recognitions sites

Answer 13

Sequences of DNA cut by restriction endonucleases

Question 14

Sticky ends

Answer 14

If recognition site cut in staggered fashion, cut ends of DNA double strand left with single strand which is a few nucleotide bases long. Nucleotides on single strand at one side of cut are complementary to those at other side.
If same restriction endonucleases used to cut DNA then all fragments produced will have ends that are complementary to one another, so end of one fragment can be joined to end of other fragment. One bases are paired up enzyme DNA ligase used to bind phosphate sugar framework of two sections of DNA.
So can combine DNA of one organism with that of any other organism.

Question 15

Insertion of DNA fragment into a vector

Answer 15

After promoter and terminator regions added, must join DNA fragment into carrying unit; vector. This transports the DNA into host cell. Most commonly used in plasmid, which usually contains genes for antibiotic resistance.
Restriction endonuclease used is same as one that cut DNA fragment so sticky ends of plasmid are complementary to sticky ends of DNA fragment. When DNA frag mixed with opened up fragments may eve incorporated into them, and DNA ligase makes joint permanent.

Question 16

Introduction of DNA into host cell

Answer 16

Must be reintroduced into bacterial cells, process called transformation.
Plasmids and bacterial cells mixed in medium contains Ca ions. Ca ions and changes in temp make bacterial membrane more permeable allowing plasmids to pass through cell surface membrane into cytoplasm.

Question 17

Why do not all bacterial cells posses DNA fragments with desired gene for desired protein

Answer 17

Only few bacterial cells take up plasmids when two are mixed.
Some plasmids will have closed up again without incorporating the DNA fragment.
Sometimes the DNA fragment ends join together to form its own plasmid.

Question 18

How to identify which bacterial cells have taken up plasmid

Answer 18

Bacteria have evolved mechanism for resisting effects of antibiotics usually by producing enzymes that break down antibiotic before it can destroy bacteria. The genes for production of these enzymes found in plasmids.
SEE CARD 1

Question 19

Polymerase chain reaction (PCR) is

Answer 19

A method of copying fragments of DNA, it’s automated so rapid and efficient

Question 20

PCR requires:

Answer 20

DNA fragment to be copied.
DNA polymerase- taq polymerase obtained from bacteria in hot springs.
Primers- short sequences of nucleotides that have a set of bases complementary to those at one end of each of two DNA fragments.
Nucleotides.
Thermocycler- computer controller machine, varies temperature.

Question 21

Polymerase chain reaction step 1 separation of DNA strand

Answer 21

DNA fragments, primers and DNA polymerase placed in vessel in thermocycler.
95 degrees.
Two strands of DNA fragments separate due to breaking of H bonds between strands

Question 22

Polymerase chain reaction step 2 Addition (annealing) of primers

Answer 22

55 degrees
Primers join (anneal) to their complementary bases at end of DNA fragment.
Primers provide starting sequences for DNA polymerase to begin copying DNA as DNA polymerase can only attach nucleotides to end of existing chain. Primers prevent two separate strands from rejoining.

Question 23

Polymerase chain reaction step 3 Synthesis of DNA

Answer 23

72 degrees.
Optimum temperature for DNA polymerase to add complementary nucleotides along dna strands. Begins at primer on both strands and adds nucleotides in sequence until it reaches end of chain.

Question 24

Advantages in vitro gene gene cloning

Answer 24

Extremely rapid, good for forensics.

Does not require living cells.

Question 25

Advantages of in vivo gene cloning

Answer 25

Particularly useful when we want to introduce a new gene into an organism, because it involves use of vectors and plasmids so gene can be delivered into diff organism (done in gene therapy).
Involves almost no risk or contamination, because restriction endo used are the same so can match sticky ends of opened up plasmid.
Very accurate.
Cuts out specific genes, so very precise.
Produces transformed bacteria that can be used to produce large quantities of gene products (i.e. medical use).

Question 26

Benefits of recombinant DNA technology

Answer 26

Microorganisms can be modified to produce antibiotics hormones etc to treat diseases and disorders. Can also be used to control pollution.
GMO plants, plant farming means plants can manufacture antibodies to toxins they produce or make antigens which when injected into humans induce natural antibody production.
GMO help prevent certain diseases e.g Golden rice.
GM animals can produce expensive drugs cheaply.
Genetic fingerprinting used in forensic science.

Question 27

Risks of recombinant DNA technology

Answer 27

Ecological consequences of releasing genetically engineered organisms into environment.
Recombinant gene may pass from organism it’s placed into to another.
GM bacteria may have antibiotic resistance marker genes, may spread resistance to harmful bacteria.
Artificial selection of ‘desired’ genes reduces the generic variety essential to evolution.
Financial consequences of developing plants in new regions.
Get into wrong hands.
Is financial cost worth it.
What if dna sample exchanged maliciously in genetic fingerprinting.
Should we tamper with genes.

Question 28

To find where particular DNA sequence is located

Answer 28

Use DNA probes and DNA hybridisation.

Determine whether someone possesses mutant allele that causes particular disorder.

Question 29

DNA probe

Answer 29

Short single stranded length of DNA that has label attached to make it easily identifiable

Question 30

Most common probes

Answer 30

Radioactively labelled probes, made of nucleotides with isotope 32P. Probe identified using an X ray film that is exposed by radioactivity.
Fluorescently labelled probes, emit light under certain conditions eg when probe bound to DNA sequence

Question 31

How do DNA probes identify particular alleles or genes

Answer 31

DNA probe is made that has base sequences that are complementary to part of base sequence of DNA that makes up allele of gene we want to find.
Double stranded DNA that is being tested so treated to separate strands.
Strands mixed with probe, which binds to complementary base sequence on one of strands. Called DNA hybridisation.
Site at which probe binds can be identified by radioactivity or fluorescence that probe emits.

Question 32

DNA hybridisation explanation

Answer 32

Takes place when section of DNA or RNA is combined with single stranded section of DNA which has complementary bases.
Before hybridisation can take place two strands of DNA must be separated, achieved by heating DNA until its double strand separates (dénaturation). When cooled complementary bases on each strand recombine (anneal) with each other to reform original strand. Strands will pair up with their partners or other complementary sections of DNA

Question 33

Process of locating specific alleles of genes

Answer 33

Determine sequence of nucleotide bases of mutant allele. Refer to genetic library or dna sequencing techniques.
Fragment of DNA produced with sequence of bases that are complimentary to mutant allele.
Multiple copies of DNA probe formed using polymerase chain reaction.
DNA probe made by attaching marker eg fluorescent dye to DNA fragment.
DNA from person suspected of having mutant allele we want to locate is heated to separate strands.
Separated strands are cooled in mix containing many of our DNA probes.
If DNA contains mutant allele one of probes is likely to bind to it because probe has base sequences that are exactly complementary to those on mutant allele.
DNA is washed clean or any unattached probes.
Remaining hybridised DNA will be fluorescently labelled with dye attached to probe.
Dye is detected by shining light into fragments causing dye to fluoresce.
SEE PG 283

Question 34

How to test for multiple different genetic disorders simultaneously

Answer 34

It is possible to fix hundreds of different DNA probes in an array (pattern) and on a glass slide. By adding a sample of DNA polymerase o the array anu complementary DNA sequences in donor DNA will bind to one or more probes. Can detect fluorescence that occurs where binding takes place.

Question 35

Another area where genetic screening can be valuable

Answer 35

Detection of oncogene, responsible for cancer. Cancers may develop as a result of mutations that prevent the tumour suppressor genes inhibiting cell division. Mutations of both allele must be present to inactivate tsg and initiate tumour.
When individuals told, they can make informed decisions about lifestyle and screen regularly.
Can inform of gene changes that predict which cancer treatments are best.
Can detect single cancer cell among millions of normal cells

Question 36

Personalised medicine

Answer 36

Genetically screening patients mean docs can determine dose of drug which will produce desired outcome. Saves money, prevents wastage. Also prevents meds that could cause harm/ false hope.
E.g. prescribing of painkillers- to function they need specific enzyme to activate them, about half pop have genes that alter function of enz so screening for presence of those genes allows dosage to be adjusted. To compensate for ways in which genes affect metabolism of painkiller.

Question 37

Genetic counselling

Answer 37

Special form of social work where advice and info are given that enable people to make personal decisions about themselves or their offspring.
Can inform of emotional, psychological, medical, social and economic consequences.

Question 38

Genetic fingerprinting relies on

Answer 38

Fact that genome of most eukaryotic organism contains many repetitive non coding bases of DNA.

Question 39

DNA bases which are non coding are known as

Answer 39

Variable number tandem repeats (VNTRS).
Different in all individuals except identical twins.
More closely related two individuals are the more similar the VNTRS will be.

Question 40

Gel electrophoresis

Answer 40

Used to separate DNA fragments according to size.
DNA fragments placed onto agar gel, voltage is applied across it.
Resistance of gel means the larger the fragment the more slowly they move.
Smaller fragments move further.
DNA fragments of different lengths separated. If DNA fragments are labelled e.g. with radioactive DNA probes the final positions in gel can be determined by placing a sheet of x-ray film over agar gel for several hours. Radioactivity from each DNA fragment exposes the film and shows where fragment is situated. Only DNA fragments up to 500 bases long can be sequenced in this way, larger genes must be cut into smaller fragments by restriction endonucleases
See card 2

Question 41

Making of genetic fingerprint

Answer 41

Extraction
Digestion 
Separation 
Hybridisation 
Development

Question 42

Extraction

Answer 42

Extract DNA by separating it from rest of cell.
As amount of DNA is usually small, quantity can be increased by using the polymerase chain reaction
See card 3a.

Question 43

Digestion

Answer 43

DNA cut into fragments using same restriction endonucleases endonucleases chosen for ability to cut close to, but not within, target DNA
See card

Question 44

Separation

Answer 44

Fragments of DNA separated according to size by gel electrophoresis under influence of electrical voltage. The gel is then immersed in alkali to separate the double strands into single strands.
See card

Question 45

Hybridisation

Answer 45

Radioactive or fluorescent DNA probes now are used to bind with VNTRs. Probes have base sequences which are complimentary to base sequences of VNTRS and bind to them under specific conditions eg temperature and pH.
Process carries out with different probes which bind to different target DNA sequences.
See card

Question 46

Development

Answer 46

X ray film put over nylon membrane. Film is exposed by radiation from radioactive probes (if using fluorescent probes positions are located visually). Because these points correspond to position of DNA fragments a series of bars is revealed. Pattern of bands is unique to every individual except identical twins.
See card

Question 47

How to interpret results of DNA fingerprinting

Answer 47

Pattern of bars of each fingerprint is passed through automated scanning machine, which calculates length of DNA fragments from bands. Does this by using data obtained by measuring distances travelled during electrophoresis by known lengths of DNA.
Odds are calculated of someone else having identical fingerprint.

Question 48

Uses of genetic fingerprinting

Answer 48

Paternity. 
Determining genetic variability in population. More closely related = closer resemblance of genetic fingerprints. 
Forensic science. 
Medical diagnosis. 
Plant and animal breeding

Question 49

Forensic science

Answer 49

Although may not have committed crime ( could be close relative or DNA sample may have been contaminated by suspect’s dna or chemicals affecting action of restriction en). Probability that someone else’s DNA might match that if suspect must be calculated. Made on assumption that DNA which produces banding patterns is randomly distributed in the community but this is not the case for some religious or ethnic groups.

Question 50

Medical diagnosis

Answer 50

Genetic fingerprints can help in diagnosing diseases such as Huntington’s disease (genetic disorder of nervous system resulting from three base sequence at one end of gene being repeated over and over again.
Genetic fingerprinting also used to identify nature of microbial infection by comparing fingerprints of microbes found in patients with that of known pathogens.

Question 51

Plant and animal breeding

Answer 51

Can be used to prevent undesirable inbreeding during breeding programs.
Can also identify plants or animals that have particular allele of desirable gene; these individuals can be selected for breeding.
Also determination of paternity in animals and therefore establishing pedigree of individual