Dna Fragmentation

Question 1

How are transcriptase enzymes used in dna fragmentation

Answer 1

Genes can be isolated and copied using reverse transcriptase enzymes:

Isolate the Eg. Islets of Langerhans cells in the pancreas that secrete insulin protein, as these have lots of mRNA in the cytoplasm for insulin production

Extract the MRNA and add reverse transcriptase which makes a single stranded complimentar copy of DNA (CDNA, complementary DNA) from the RNA template

Add DNA polymerase to make this into a double DNA strand.

Question 2

How can genes be isolated and removed using endonuclease enzymes

Answer 2

Genes can be isolated and removed using restriction endonuclease enzymes:

Restriction endonucleases recognize and cut recognition sequences which are specific palindromic DNA sequences

The shape of the active site is complementary to the recognition sequence

This leaves sticky ends: small tails of unpaired bases. These
can easily bind (anneal) to any other DNA fragment cut using the same restriction
enzyme

Question 3

Gene machine in synthesising genes from scratch

Answer 3

Genes can be synthesised from scratch using a ‘Gene machine

Design a new sequence to make the desired amino acid sequence of the new protein

Attach the first nucleotide to a bead then add others step by step

Question 4

DNA amplification in vitro

Answer 4

DNA Amplification in vitro by Polymerase Chain Reaction
(PCR):

• Produces a large number of copies of any specific fragments of DNA

This enables tests to be carried out on small/old samples

• Repeat about 25 times to get millions of DNA copies

Step 1 Denaturation: Heat to 95°C breaks hydrogen bonds separate DNA strands which become templates for new complimentary strands

Step 2 Annealing: Cooling to 55°C causes primers to attach DNA template strands at complimentary sequence as hydrogen bonds reform

Step 3 Synthesis of new dna: raise temperature to 70 degrees Celsius, optimum for DNA polymerase which attaches to primer and adds new bases complimentary to the template

Question 5

DNA amplification in Vivo by transforming host cells

Answer 5

1. ISOLATE the human gene coding for the insulin protein from Islets of Langerhans cells in the pancreas using a gene probe.
   Cut the DNA with restriction endonucleases, which cut DNA at
   specific base sequences (restriction site) to produce fragments with staggered ‘sticky ends’: single stranded exposed unpaired bases
2. INSERT the gene into a vector eg. An E.coli bacterial plasmid (additional loop of DNA). The plasmid DNA is cut using the same restriction endonuclease that is used to produce the same sticky ends so the insulin gene will fit in as the unpaired bases are complimentary. DNA ligase is used to ‘anneal’ (splice/ioin)
   the donor and vector DNA. This DNA is now called recombinant DNA
3. UPTAKE of the new plasmids into E.coli bacteria

• Only about 1% of E.coli incorporate the insulin gene. Use marker genes to identify them. Eg radioactive labeling, or a
gene for antibiotic

4. CULTURE the E.coli to produce many clones containing copies of the insulin gene which produced lots of insulin protein product which is separated and purified

Question 6

Genetic fingerprinting

Answer 6

1. A sample of DNA is obtained e.g. from blood, saliva.
2. PCR is used to make many copies of the area of DNA that contains VNTRS (primers
   are used to bind to either side of the repeat so that all of it is amplified)

3.DNA fragments of variable length are produced- depending on the number of repeats

4. A fluorescent tag is added to DNA
   sample weils fragments so they can be viewed under UV light
5. DNA fragments undergo
   electrophoresis
6. DNA fragments are viewed as bands under UV light- the genetic fingerprint.

Question 7

Genetic finger printing process

Answer 7

1. A sample of DNA is obtained e.g. from blood, saliva.
2. PCR is used to make many copies of the area of DNA that contains VNTRs (primers are used to bind to either side of the Power supply
   repeat so that all of it is amplified)
3. DNA fragments of variable length are produced - depending on number of repeats
4. A fluorescent tag is added to DNA
   sample wells
   fragments so they can be viewed under UV light
5. DNA fragments undergo electrophoresis

• DNA is placed into well in a slab of gel covered in buffer solution that
buffer solution that conducts electricity

• An electrical current is passed through the well

• DNA fragments are negatively charged so move towards positive electrode

• Smaller DNA fragments move faster and travel further through the gel so the fragments separate according to size.

6. DNA fragments are viewed as bands under UV light- the genetic fingerprint.

Question 8

Probes for identifying genetic conditions

Answer 8

DNA probes are short strands of DNA that have a specific sequence which is complementary to part of a target allele.

This means that it will bind or hybridise to the target allele

Can be used to locate specific alleles of genes e.g. to diagnose genetic disorders.

Can also be used to identify health risks e.g. to cancer and determine how patients will respond to drugs and inform personalised drug treatment.

Question 9

VNTRS in genetic fingerprinting

Answer 9

VNTRs (variable number tandem repeats) are non-coding sequences of DNA in a genome.

They repeat a different number of times. E.g. CAT could be CATCATCATCATG or
CATGCATG

The probability of two individuals have the same VNTRs is very low.

Electrophoresis separates DNA fragments to make a genetic fingerprint

Question 10

Markscheme : describe how genetic fingerprinting may be carried out in a sample of panda DNA

Answer 10

1.DNA is cut

2.using restriction enzyme

3. Use electrophoresis
4. Separates according to length/mass
5. Southern blotting / transfer to (nylon) membrane
6. Make single strand
7. Apply probe
8. Radioactive/ fluorescent