Gene Sequencing

Question 1

What is recombinant DNA technology

Answer 1

Cell having two or more sources of DNA by isolating fragments of DNA from gene of interest and inserting this DNA into another organism

Question 2

Universal

Answer 2

All the same triplets code for all the same amino acids in all organisms

Question 3

Degenerate

Answer 3

More than one triplet codes for an amino acid

Question 4

Overlapping

Answer 4

Each base is only read once

Question 5

Five steps of recombinant DNA technology (IITIG)

Answer 5

Isolation of genes
Insertion
Transformation
Identification
Growth

Question 6

Three ways of isolating DNA fragments

Answer 6

Reverse transcriptase= conversion of mRNA to complementary DNA
Using restriction endonucleases = cut fragments containing desired gene from DNA
Gene machine = creating the gene

Question 7

Process of using reverse transcripts in isolation

Answer 7

1. mRNA selected from cell that produce proteins of interest in abundant numbers
   mRNA template is mixed with DNA nucleotides and reverse transcriptase
2. Free DNA nucleotides by a single stranded mRNA template via complementary base pairing.
3. Reverse transcription joins DNA nucleotides together to form single stranded C DNA molecule.
4. DNA polymerase is required to make C DNA double stranded.

Question 8

Using restriction endonuclease for isolation

Answer 8

1. There are enzymes that hydrolyse DNA at specific region base sequences.
2. Different restriction endonuclease hydrolysed DNA are different specific Regions sequences because the shape of the sequence of complimentary to the enzymes active site
3. DNA samples incubated with specific restriction nucleases
4. Hydrolyse the DNA into fragments
5. If the target gene has recognition sequences before and after the target gene, the fragments will contain the desire gene , into blunt or sticky ends
6. Possible to add promoter region to stimulate our newly or terminator region to stop transcription.

Question 9

The gene machine isolation

Answer 9

1. Desired nucleotide sequence fed into a computer.
2. Examine protein to identify amino acid sequence and from that the mRNA and DNA sequence.
3. Entered into computer to check if DNA being created is safe and ethical to produce
4. Computer can create small sections of overlapping single strands of nucleotide that make up the gene called oligonucleotide

Question 10

Advantages/ disadvantages of using gene machine

Answer 10

Faster to use gene machine than all the enzyme catalysed reactions with reverse transcriptase
Need to know amino acid sequence

Question 11

Advantages/ disadvantages of reverse transcripts

Answer 11

Endonuclease isolate gene from human DNA

Human gene contains introns

Bacteria cannot remove introns by splicing them

Reverse transcriptase/genemachine produces gene without introns.

mRNA much easier to obtain

Question 12

Advantages/disadvantages of restriction endonucleases

Answer 12

Contains Intron
Sticky and some DNA fragments make it easier to insert to make recombinant DNA

Question 13

Describe the process of inserting a gene into a plasmid

Answer 13

• cut plasmid and gene with same restriction endonuclease to produce ‘sticky ends’
• both have complementary stick ends that can anneal by hydrogen bonding between complementary base pairs
• mix with DNA ligase to join sticky end parts of gene and plasmid with phosphodiester bonds

Question 14

why do scientists aim to insert a vector (plasmid with required gene) into:
1. cytoplasm or circular DNA of bacteria
But
2. nuclear DNA of humans.

Answer 14

Bacteria divide by binary fission that copies plasmids and circular chromosome that contain desired gene to produce genetically identical cells
But
Human cells divide by mitosis which will only copy desired gene if it is in nuclear DNA to produce genetically identical cells.

Question 15

When inserting a vector (plasmid containing gene) into eukaryotes, why do scientists inject into gametes/zygotes and not somatic (body) cells

Answer 15

Zygote cell is totipotent so divide by mitosis and differentiate into all cells of organism.
Therefore gene will be in cell that will transcribe gene into mRNA and translate mRNA into named protein

Question 16

Explain the purpose of attaching a gene such as GFP jelly fish gene alongside the gene of interest into the vector that is inserted into embryo/zygote

Answer 16

Not all cells will successfully take up the plasmid
GFP gene is a marker gene to show transformed cells and what cells are expressing the desired gene.
As only cells/embryos will show fluorescence and glow under UV light

Question 17

Describe the polymerase chain reaction (5 marker)

Answer 17

1. Heat DNA (94-98 degrees) to separate strands by breaking hydrogen bonds.
2. Add primers (short ssDNA sequences) that has specific base sequence that is complementary to start of allele (whatever dna is to be copied).
3. Add free DNA nucleotides.
4. Cool temperature (50-64 degrees) to allow binding of
   primer and nucleotides to DNA by complementary base ( DNA hybridzation )
   pairing with H bonds.
5. Increase temperature (72-80 degrees) to allow heat
   stable taq DNA polymerase to join adjacent DNA nucleotides with phosphodiester bonds in condensation reactions to produce complementary DNA
6. Repeat cycles many times

Question 18

Primer

Answer 18

Short single strand of DNA sequences which have a specific base sequence that is complementary to the start of allele

Question 19

DNA probe

Answer 19

Short single strand of DNA sequences which have a specific base sequence that is complementary to known sequence of DNA

Question 20

explain why you may make DNA probe dyed/ radioactive

Answer 20

DNA is invisible so radioactive nucleotide/dye gives out light to allow for detection of the location of gene on the chromosome the probe is attached to

Question 21

How do you screen DNA for multiple DNA sequences of interest/genes

Answer 21

1. amplification: Use PCR to replicate many copies of DNA
2. Cut DNA using restriction endonucleases
3. Separate DNA fragments using gel electrophoresis
4. Add a labelled DNA probe that binds by DNA hybridisation
5. Shine a UV light to identify DNA of interest//genes by fluorescence
   or
6. Compare positions of bands to known DNA sample with all genes/DNA sequences of interest
   Or
   
   5. See appearance of gel after X-ray film is taken for identification.

Question 22

Who’s the father? Explain

Answer 22

child has 50% of DNA from mum and 50% from dad.
So bands on child’s fingerprint that are not given from mum, must be from father.

Question 23

Describe how genetic fingerprinting can be carried out on a sample of DNA and why it can be used to identify a person.

Answer 23

1. ExtractDNAfromsampleandcutusingrestrictionendonucleaseto make blunt end fragments.
2. CutmustleaveVNTRs(variablenumbertandemrepeats)intact
3. SeparateDNAfragmentsaccordingtolengthusingelectrophoresis
4. Byplacingsampleintowellongelandpassingelectriccurrentthrough.
5. UsealkalinesolutiontomakeDNAsinglestranded
6. Use southern blotting to transfer to nylon membrane.
7. DNA hybridisation with added radioactive DNA probe and target fragment.
8. Usex-rayfilmtosee
9. Patternuniquetoindividual

Question 24

Name and explain factors may slow down the process of obtaining a genetic fingerprint?

Answer 24

1. ifonlySmallamountofDNAobtained->needsPCRforamplification-> PCR increases amount of DNA so enough available for genetic fingerprinting.
2. IfDNAsampleiscontaminatedwithotherDNApresent->needtoidentify required DNA from rest and then amplify using PCR.