8 Recombinant DNA tech

Question 1

recombinant dna technology

Answer 1

Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism.

Question 2

how is it possible that the dna of one organism is accepted by another species which functions normally when it is transferred?

Answer 2

the genetic code is the same in all organisms

it is universal and can be used by all living organisms

Question 3

what are the stages of the process of making a protein using the dna technology of gene transfer and cloning?

Answer 3

1. isolation- of the dna fragments that have the gene for the desired protein
2. insertion- of the dna fragment into a vector
3. transformation- that is, the transfer of dna into suitable host cells
4. identification- of the host cells that have successfully taken up the gene by use of gene markers
5. growth/cloning- of the population of host cells

Question 4

what are the several methods of producing dna fragments?

Answer 4

• conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
• using restriction endonucleases to cut a fragment containing the desired gene from DNA
• creating the gene in a ‘gene machine’.

Question 5

using reverse transcriptase

Answer 5

• a cell that readily produces the protein is selected (e.g. the B-cells of the islets of Langerhans from the pancreas are used to insulin)
• these cells have large quantities of the relevant mRNA, which is therefore more easily extracted
• reverse transcriptase is then used to make DNA from RNA. this DNA is known as complementary DNA (cDNA) as it is made up of the nucleotides that are complementary to the mRNA
• to make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. this double strand of DNA is the required gene

Question 6

using restriction endonucleases

Answer 6

RE are enzymes that cut up viral dna
many kinds of RE, each one cuts a dna double strand at a specific sequence
sometimes, this cut occurs two opposite base pairs, leaving two straight edges known as blunt ends
other RE cut dna in a staggered fashion, leaving an uneven cut in which each strand of the dna has exposed, unpaired bases

Question 7

the gene machine

Answer 7

• the desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce. the AA sequence of this protein is determined. from this, the mRNA codons are looked up and the complementary dna triplets are worked out.
• the desired sequence of nucleotide bases for the gene is fed into a computer
• the sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements
• the computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene
• in an automated process, each of the oligonucleotides is assembled by adding one at a time in the required sequence
• the oligonucleotides are joined together to make a gene. this gene doesn’t have introns. the gene is replicated using the polymerase chain reaction.
• the polymerase chain reaction also constructs the complementary stand of nucleotides to make the required double stranded gene. it then multiplies this gene many times to give numerous copies.
• using sticky ends the gene can then be inserted into a bacterial plasmid. this acts as a vector for the gene allowing it to be stored, clones or transferred to other organisms.
• the genes are checked using standard sequencing techniques and those with errors are rejected

Question 8

advantages of the gene machine

Answer 8

any sequence of nucleotides can be produced, in a 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 9

in what ways can fragments of dna be cloned so there is a sufficient quantity for medical and commercial use?

Answer 9

in vivo- by transferring the fragments to a host cell using a vector
in vitro- using the polymerase chain reaction

Question 10

importance of sticky ends

Answer 10

sequences of dna that are cut by restriction endonucleases are called recognition sites
if the recognition site is cut in a staggered fashion, the cut ends of the dna double strand are left with a single strand at which is a few nucleotide bases long.
the nucleotides on the single strand at one side are complementary to those at the other side
if the same RE is used to cut dna, then all the fragments produced will have ends complementary to one another.
meaning that the single-stranded end of any one fragment can be joined to any other fragment
once the complementary bases of two sticky ends have paired up, an enzyme called dna ligase is used to bind the phosphate-sugar framework of the two sections of dna and so unite them as one
sticky ends are important, provided the same RE is used, we can combine the dna of one organism with that of any other organism

Question 11

preparing the dna fragment for insertion- in vivo cloning

Answer 11

• the preparation of the dna fragment involves the addition of extra lengths of dna
• for the transcription of any gene to take place, the enzyme that synthesises mRNA (RNA polymerase) must attach to the dna near a gene
• the binding site for RNA polymerase is a region of dna, known as a promoter
• the nucleotide bases of the promoter attach both RNA polymerase and transcription factors and so begin the process of transcription
• another region releases RNA polymerase and ends transcription
• this region of dna is called a terminator

Question 12

insertion of dna fragment into a vector- in vivo cloning

Answer 12

• once the dna fragment has been prepared for insertion, it is joint to a carrying unit, known as a vector
• this vector is used to transport the dna into the host cell
• there are diff types of vector but the most commonly used is the plasmid
• plasmids are circular lengths of dna, found in bacteria which are separate from the main bacterial dna
• plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop
• the RE used is the same one that cut out the dna fragment, ensuring that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the dna fragment
• when the dna fragments are mixed with the opened-up plasmids, they become incorporated into them
• where they are incorporated, the join is made permanent using the enzyme dna ligase

Question 13

introduction of dna into host cells- in vivo cloning

Answer 13

• once the dna has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells. this process is called transformation
• transformation involves the plasmids and bacterial cells being mixed tog in a medium containing calcium ions
• the calcium ions, and changes in temp, make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm
• however, not all the bacterial cells will possess the dna fragments with the desired gene for the desired protein

Question 14

why don’t all the bacterial cells possess the dna fragments with the gene for the desired protein?- in vivo cloning

Answer 14

• only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together
• 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 15

gene markers- in vivo cloning

Answer 15

there are a number of different ways of using marker genes to identify whether a gene has been taken up by bacterial cells
they all involve using a second, separate gene on the plasmid
this second gene is easily identifiable, e.g. it may be resistant to an antibiotic, make a fluorescent protein that is easily seen, or produce an enzyme whose action can be identified

Question 16

antibiotic-resistance marker genes- in vivo cloning

Answer 16

to identify those cells with plasmids that have taken up the new gene we use a technique called replica plating
this process uses the other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene
as this gene has been cut, it will no longer be able to produce the enzyme that breaks down a specific antibiotic, so we can therefore identify these bacteria by growing them on a culture that contains the antibiotic
the treatment with the antibiotic will destroy the cells that contain the required gene, so by using a technique called replica plating, it is possible to identify living colonies of bacteria containing the required gene

Question 17

fluorescent markers- in vivo cloning

Answer 17

view cells under microscope

retain those that do not fluoresce

Question 18

dna probes

Answer 18

a short, single-stranded length of dna that has some sort of label attached that makes it easily identifiable
the two most commonly used probes are:
-radioactively labelled probes
-fluorescently labelled probes

Question 19

radioactively labelled probes

Answer 19

made up of nucleotides with the isotope 32P

the probe is identified using an x ray film that is exposed by radioactivity

Question 20

fluorescently labelled probes

Answer 20

emit light (fluoresce) under certain conditions, e.g. when the probe has bound to the target dna sequence

Question 21

in what way are dna probes used to identify particular alleles of genes?

Answer 21

• a dna probe is made that has base sequences that are complementary to part of the base sequence of the dna that makes up the allele of the gene that we want to find
• the double stranded dna that is being tested is treated to separate its two strands
• the separated dna strands are mixed with the probe, which binds to the complementary base sequence on one of the strands. this is known as dna hybridisation
• the site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits

Question 22

dna hybridisation

Answer 22

• takes place when a section of dna or rna is combined with a single-stranded section of dna which has complementary bases
• before hybridisation can take place, the two strands of the dna molecule must be separated
• this is achieved by heating dna until its double strand separates into its two complementary single strands (denaturation)
• when cooled, the complementary bases on each strand recombine (anneal) with each other to reform the original double strand
• given sufficient rime, all strands in a mixture of dna will pair up with their partners
• if, however, other complementary sections of dna are present in the mixture as the dna cools, these are just as likely to anneal with one of the separated dna strands as the two strands are with one another

Question 23

locating specific alleles of genes purpose

Answer 23

• using dna probes and dna hybridisation, it is possible to locate a specific allele of a gene
• e.g. we may wish to determine whether someone possesses a mutant allele that causes a particular genetic disorder

Question 24

process of locating specific alleles of genes

Answer 24

• first determine the sequence of nucleotide bases the mutant allele we are trying to locate. this can be achieved using dna sequencing techniques. however, we now have extensive genetic libraries that store the base sequences of most genetic diseases and so we can simply refer to these to obtain the sequence
• a fragment of dna is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate
• multiple copies of our dna probe are formed using the polymerase chain reaction
• a dna probe is made by attaching a marker, e.g. a fluorescent dye, to the dna fragment
• dna from the person suspected of having the mutant allele we want to locate is heated to separate its two strands
• the separated strands are cooled in a mixture containing many of our dna probes
• if the dna contains the mutant allele, one of our probes is likely to bind to it as the probe has base sequences that are exactly complementary to those on the mutant allele
• the dna is washed clean of any unattached probes
• the remaining hybridised dna will now be fluorescently labelled with the dye attached to the probe
• the dye is detected by shining light onto the fragments causing the dye to fluoresce which can be seen using a special microscope

Question 25

genetic screening

Answer 25

• it is important to screen individuals who may carry a mutant allele
• such individuals often have a family history of a disease
• screening can determine the probabilities of a couple having offspring with a genetic disorder
• it is possible to fix hundreds of different dna probes in an array on a glass slide. by adding a sample of dna to the array, any complementary dna sequences in the donor dna will bind to one or more probes.
• in this way it is possible to test simultaneously for many different genetic disorders by detecting fluorescence that occurs where binding has taken place
• if a mutated gene is detected, individuals who are at a greater risk of cancer can then make informed decisions about their lifestyle and future treatment.

Question 26

personalised medicine

Answer 26

• genetic screening allows doctors to provide advice and health care based on sn individual’s genotype
• some people’s genes can mean that a particular drug may be either more or less effective in treating a condition
• by genetically screening patients, doctors can determine, more exactly, the dose of a drug which will produce the desired outcome
• this can save money that would otherwise be wasted on overprescribing the drug

Question 27

genetic counselling

Answer 27

• a special form of social work, where advice and information are given that enable people to make personal decisions about themselves or their offspring
• one important aspect is to research the family history of an inherited disease and to advise parents on the likelihood of it arising in their children
• genetic counselling is closely linked to genetic screening and the screening results provide the genetic counsellor with a basis for informed discussion

Question 28

genetic fingerprinting

Answer 28

• technique relies on the fact that the genome of most eukaryotic organisms contains many repetitive, non-coding bases of dna
• dna bases which are non-coding are known as variable number tandem repeats (VNTRs)
• for every individual the number and length of VNTRs has a unique pattern
• they are different in all individuals except identical twins, and the probability of two individuals having identical sequences of these VNTRs is extremely small
• however, the more closely related two individuals are, the more similar the VNTRs will be

Question 29

gel electrophoresis

Answer 29

-used to separate dna fragments according to their size
-the dna fragments are placed on to an agar gel and a voltage is supplied across it
-the resistance of the gel means that the larger the fragments, the more slowly they move
therefore, over a fixed period, the smaller fragments move further than the larger ones
-in this way dna fragments of diff lengths are separated
-if the dna fragments are labelled, e.g. with radioactive dna probes, their final positions in the gel can be determined by placing a sheet of x-ray film over the agar gel for several hours
-the radioactivity from each dna fragment exposes the film and shows where the fragment is situated on the gel

Question 30

what are the main stages of the making of a genetic fingerprint?

Answer 30

extraction
digestion
separation
hybridisation
development

Question 31

extraction- genetic fingerprinting

Answer 31

even the tiniest sample of animal tissue, such as a drop of blood or a hair root, is enough to give a genetic fingerprint
whatever the sample, the first stage is to extract the dna by separating it from the rest of the cell
as the amount of dna is usually small, its quantity can be increased by using the polymerase chain reaction

Question 32

digestion- genetic fingerprinting

Answer 32

the dna is then cut into fragments using the same restriction endonucleases
the endonucleases are chosen for their ability to cut close to, but not within, the target dna

Question 33

separation- genetic fingerprinting

Answer 33

the fragments of dna are next separated according to size by gel electrophoresis under the influence of an electrical voltage
the gel is then immersed in alkali in order to separate the double strands into single strands

Question 34

hybridisation- genetic fingerprinting

Answer 34

radioactive or fluorescent dna probes are now used to bind with VNTRs
the probes have base sequences which are complementary to the base sequences of the VNTRs, and bind to them under specific conditions, such as temp and pH
the process is carried out with different probes, which bind to different dna sequences

Question 35

development- genetic fingerprinting

Answer 35

finally, an x-ray film is put over the nylon membrane
the film is exposed by radiation from the radioactive probes
or if using fluorescent probes, the positions are located visually
because these points correspond to the position of the dna fragments as separated during electrophoresis, a series of bars is revealed
the pattern of the bands is unique to every individual except identical twins

Question 36

interpreting the results of genetic fingerprinting

Answer 36

dna fingerprints from two samples, e.g., the blood found at the scene of a crime and from a suspect, are visually checked
if there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of the dna fragments from the bands
it does this using dna obtained by measuring the distances travelled during electrophoresis by known lengths of dna
finally, the odds are calculated of someone else and having identical fingerprint
the closer the match between the two patterns, the greater the probability that the two sets of dna have come from the same person

Question 37

uses of dna fingerprinting

Answer 37

genetic relationships and variety
forensic science
medical diagnosis
plant and animal breeding

Question 38

genetic relationships and variability- uses of dna fingerprinting

Answer 38

dna fingerprinting can be used to help resolve questions of paternity
individuals inherit half their genetic material from their mother and half from their father
therefore, each band on a dna fingerprint of an individual should have a corresponding band in one of the parents’ dna fingerprint
this can be used to establish whether someone is the genetic father of a child
GF is also useful in determining genetic variability within a population
a population whose members have very similar GF has little genetic diversity

Question 39

forensic science- uses of dna fingerprinting

Answer 39

dna is often left at the scene of a crime, e.g. blood, semen, hair.
GF can establish whether a person is likely to have been present at the scene of a crime, although this does not prove that they actually carried out the crime
the probability that someone else’s dna might match that of the suspect has to be calculated
this calculation is based on the assumption that the dna which produces the banding patterns is randomly distributed in the community

Question 40

what possible explanations need to be investigated about dna found at crime scenes in forensic science

Answer 40

• the dna may have been left on some other, innocent occasion
• the dna may belong to a very close relative
• the dna sample may have been contaminated after the crime, either by the suspect’s dna or by chemicals that affected the action of the restriction endonucleases used in preparing the fingerprint

Question 41

medical diagnosis- uses of dna fingerprinting

Answer 41

-GF can help in diagnosing diseases such as Huntington’s disease
a sample of dna from a person with the allele for HD can be cut with restriction endonucleases and a dna fingerprint prepared
this can then be matched with fingerprints of people with various forms of the disease and those without the disease
in this way, the probability of developing symptoms and when can be determines
-GF are also used to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with known pathogens

Question 42

plant and animal breeding- uses of dna fingerprinting

Answer 42

-GF can be used to prevent undesirable inbreeding during breeding programmes on farms or in zoos
-it can also identify plants or animals that have a particular allele of a desirable gene
individuals with this allele can be selected for breeding in order to increase the probability of their offspring having the characteristic that it produces
-another application is the determination of paternity in animals and thus establishing the pedigree (family tree) of an individual