recombinant DNA technology

Question 1

Explain how complementary DNA is made using reverse transcriptase

Answer 1

-Strand of mRNA from the cells that produce the desired protein
-mRNA acts as a template for production of a single stranded complementary copy of DNA (cDNA) using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with an enzyme
-Double stranded DNA is formed on the template of the cDNA using DNA polymerase
-This is the gene for the protein

Question 2

Explain how restriction endonucleases are used to cut DNA into fragments

Answer 2

-Restriction endonucleases cut DNA at specific base sequence (recognition site)
-breaking phosphodiester bonds
-This produces ‘sticky ends’ (short unpaired sequence of bases)
-Sticky ends will be complementary to the sicky ends on other DNA cut with the same restriction endonuclease

Question 3

Define recombinant DNA technology

Answer 3

process by which genes are altered, manipulated and transferred between organisms

Question 4

What is recombinant DNA

Answer 4

DNA of two different organisms that has been combined

Question 5

Explain why it is possible to transfer DNA between organisms and for proteins to still be produced

Answer 5

the genetic code and protein production process are universal

Question 6

Explain how the gene machine is used to produce DNA fragments

Answer 6

-Desired protein
-Amino acid sequence
-mRNA sequence/triplets
-DNA sequence
-Nucleotide base sequence into computer for Biosafety/biosecurity/international standards/ethics check
-Computer designs oligonucleotides (small, overlapping single strand nucleotides)
-Oligonucleotides assembled one nucleotide at a time (automated)
-Oligonucleotides joined to form gene (all coding DNA. Single strand)
-PCR replicates gene (makes complemetary strand for DNA first)
-Gene into plasmid using sticky ends (vector for storage/cloning/transfer)
Genes check using sequencing techniques

Question 7

State the stages in making a protein using DNA technology

Answer 7

isolation, insertion, transformation, identification, growth/cloning

Question 8

State 3 ways to isolate a fragment of DNA

Answer 8

reverse transcriptase, restriction endonucleases, gene machine

Question 9

What is the function of reverse transcriptase?

Answer 9

to produce DNA from RNA

Question 10

Explain how DNA is made using reverse transcriptase

Answer 10

isolate mRNA –> reverse transcriptase makes single strand of cDNA complementary to mRNA –> isolate single strand of cDNA by hydrolysis –> DNA polymerase forms double stranded DNA

Question 11

What is a restriction endonuclease?

Answer 11

enzyme that cuts a strand of DNA at a specific base sequence (recognition site)

Question 12

What is a sticky end?

Answer 12

short sequence of unpaired bases left after DNA is cut with restriction endonuclease

Question 13

Describe the purpose of the gene machine

Answer 13

to produce a double stranded gene from a know base sequence

Question 14

Describe how a DNA base sequence can be determined from a protein

Answer 14

protein –> amino acid sequence –> mRNA codons –> complementary DNA base sequence

Question 15

What is a a DNA base sequence checked for before production in a gene machine?

Answer 15

biosafety and biosecurity

Question 16

Name the short sections of DNA produced in the gene machine

Answer 16

oligonucleotides

Question 17

How is the order of the oligonucleotides in the gene determined?

Answer 17

the sections overlap

Question 18

State two ways to clone genes

Answer 18

-in vivo
-in vitro

Question 19

Explain why it is important to use the same restriction endonuclease to cut DNA from different organisms that you wish to combine

Answer 19

the restriction endonuclease will make a specific sticky end and this must be complementary to that on the other piece of DNA

Question 20

Name the enzyme used to to join the sugar phosphate backbone of recombinant DNA

Answer 20

DNA ligase

Question 21

Describe how to prepare a DNA fragment for insertion

Answer 21

attach a promotor (for RNA polymerase to bind to) and a terminator (to stop transcription)

Question 22

Explain the importance of sticky ends

Answer 22

Short unpaired sequence of bases produced when DNA is cut by a restriction endonuclease. Allow us to bind DNA from one organism to another provided the same restriction endonuclease is used

Question 23

Explain how a DNA fragment can be inserted into a vector

Answer 23

-Target DNA and plasmid cut with the same restriction endonuclease so have complementary sticky ends which base pair.
-Phosphodiester bonds formed using DNA helicase

Question 24

Explain how the DNA of the vector is introduced into host cells

Answer 24

-Transformation
-Calcium ions and change in temperature are required to make cell walls more permeable

Question 25

Describe the nature of gene markers and explain how they work

Answer 25

-Marker genes are genes in a plasmid that code for antibiotic resistance/fluorescent protein
They can be used to detect whether:
a) a bacterium has taken up a plasmid (bacteria containing the plasmid will be resistant to antibiotic/will glow)
b) the plasmid has taken up the desired gene (desired gene inserted into antibiotic resistance/fluorescence/enzyme gene - bacteria that are not resistant to antibiotic/do not glow/do not catalyse reaction contain the desired gene)

Question 26

Describe the polymerase chain reaction

Answer 26

In vitro gene cloning. Automated process of copying fragments of DNA

Question 27

Explain how the polymerase chain reaction is carried out

Answer 27

-Requires DNA fragment, DNA polymerase, (DNA) nucleotides and primers;
-Heat to 95 °C to break hydrogen bonds (and separate strands);
-Reduce temperature to 55 °C so primers bind (anneal) to DNA at their complementary bases.
-This provides a starting point for DNA polymerase to bind;
-Increase temperature to 72 °C, optimum temperature for DNA polymerase to join nucleotides;”

Question 28

Summarise the advantages of in vivo and in vitro cloning

Answer 28

In vitro:
-Rapid
-Does not require living cells
In vivo:
-Useful to introduce genes into other organisms
-No contamination
-Accurate
-Cuts out specific genes”

Question 29

Describe what DNA probes are and explain how they work

Answer 29

DNA probe is a short single strand of DNA labelled with either a radioactive or fluorescent marker, with bases complementary with required gene;
-Used to identify specific alleles of a gene by binding a detecting”

Question 30

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

Answer 30

-DNA hybridisation: when a section of DNA or RNA binds with a single stranded section of DNA with complementary bases.
-To locate gene:
-Make DNA sample single stranded
-The probe will bind to specific gene;
-Use autoradiography/x ray film/detect fluorescence to show the bound probe;”

Question 31

Describe the use of labelled DNA probes to screen for heritable conditions or health risks

Answer 31

DNA probes can be used to identify alleles of genetic disorders or that predispose people to diseases

Question 32

Consider the use of genetic screening in genetic counselling

Answer 32

Genetic screening allows people to make informed decisions during genetic counselling about themselves or their offspring

Question 33

Describe what genetic fingerprinting is

Answer 33

Process that relies on the fact that the DNA of individuals is unique and contains repetitive, non coding bases called VNTR’s (variable number tandem repeats)

Question 34

Explain the technique of gel electrophoresis

Answer 34

-Used to separate fragments of DNA in size order
-DNA fragments on agar gel with voltage across it.
-Negatively charged DNA moves towards the anode (positive)
-Larger the fragment the slower the movement
-Therefore less distance travelled in a fixed time
-Fragments labelled with DNA probes
-Determine final position on gel by applying x ray film on the gel
-Radioactivity from the DNA exposes the film and maps the fragments”

Question 35

Explain how genetic fingerprinting is carried out

Answer 35

-Extraction- extract DNA from sample
-Digestion - Restriction endonucleases cut DNA into fragments
-Separation - Separate fragments using gel electrophoresis and transfer from gel to nylon membrane
-Hybridisation - Add DNA probes to label DNA fragments
-Development - Nylon membrane with DNA fragments placed on X ray film, development of the film reveals dark bands where the probes have attached”

Question 36

Explain how the results of genetic fingerprinting are interpreted

Answer 36

-Visually compare samples and if similar use automated scanning machine which which calculates and compares DNA fragment length based on distance travelled of known samples. -Closer the match in sample patterns the more likely the samples were from the same person

Question 37

Consider the uses of genetic fingerprinting

Answer 37

-Genetic relationships - paternity case resolution, bands on children’s genetic fingerprint will each match with one of their parents
-Genetic variability - within populations, similar genetic fingerprints means little genetic diversity
-Forensic science - DNA at the scene of a crime can be analysed to determine someone’s presence at the crime scene
-Medical diagnosis - compare genetic fingerprints of patients with those of known cases
-Plant and animal breeding - prevent inbreeding in breeding programmes or identify desirable genes