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2.2.8 Studying Whole Genomes
· DNA Profiling (Fingerprinting) used in forensic crime scene analysis and paternity/maternity testing.
· Genomic Sequencing + Comparative Genome Mapping used in research into the function of genes + DNA sequences.
· Genetic Engineering used in the production of chemicals + genetically modified organisms.
· Gene Therapy used to treat conditions like cystic fibrosis.
The Genomic Age
· The DNA of all organisms contains sections (genes) which code from production of proteins this coding DNA forms only a
small part of the DNA found in an organism.
· Much DNA is non-coding DNA or junk DNA.
· Genomics is the study of the whole set of genetic information in the form of the DNA base sequences that occur in the cells
of organisms of a particular species. It aims to map the whole genome of an increasing number of organisms.
Sequencing the Genome of an Organism
1. Genomes are mapped to identify which part of the genome they have come from.
2. Samples of the genome are sheared (mechanically broken) into smaller sections.
3. These sections are placed into separate bacterial artificial chromosomes (BACs) + transferred to E.coli cells. As the cells
grow, many copies of the sections are produced these cells are clone libraries.
In order to sequence a BAC section:
1. Cells containing specific BACs are taken + cultured. The DNA is extracted from the cells + restriction enzymes are used to cut it
into smaller fragments.
2. The fragments are separated using a process known as electrophoresis.
3. Each fragment is sequenced using an automated process.
4. Computer programmes then compare overlapping regions from the cuts made by different restriction enzymes to reassemble
the whole BAC segment sequence.…read more
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2.2.9 DNA Manipulation Separating + Probing
· Electrophoresis is used to separate DNA fragments based on their size accurate enough to separate fragments that are
different by only one base in length.
· Widely used in gene technology to separate DNA fragments for identification + analysis.
· A gel plate or slab containing agarose (a type of sugar) is covered in buffer solution. Electrodes are attached to each end of
the gel so a current can be passed through it.
· The separation of strands of different lengths occurs because longer strands of DNA get caught up in the agarose gel + are
slowed, whereas shorter strands can move more quickly through the gel.
1. DNA samples are treated with restriction enzymes to cut them into fragments.
2. DNA samples are placed into wells cut into one end of the gel.
3. The gel is immersed in a tank of buffer solution + an electric current is passed through the solution for a fixed period of time.
4. DNA is negatively charged it is attracted to the +ve electrode so DNA fragments diffuse through the gel towards the +ve end.
5. Shorter lengths of DNA move further the position of the fragments can be shown by using a dye.
· The fragments may be lifted from the gel for further analysis Southern blotting. A nylon sheet is placed over the gel,
covered in paper towels, pressed + left overnight. The DNA fragments are transferred to the sheet + can be analysed. The
fragments are made visible by a radioactive marker.…read more
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· A DNA probe is a short single-stranded piece of DNA that is complementary to a section of the DNA being investigated.
· The probe is labelled in one of two ways:
Using a radioactive marker so the location can be revealed by exposure to photographic film.
Using a fluorescent marker that emits a colour on exposure to UV light.
· Copies of the probe can be added to any sample of DNA fragments as they are single stranded so they will bind to any
fragment where a complementary base sequence is present this binding is called annealing.
· Probes are useful in locating specific sequences:
To locate a specific desired gene.
To identify the same gene on a variety of different genomes.
To identify the presence/absence of an allele.…read more
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2.2.10 Sequencing + Copying DNA
The Polymerase Chain Reaction (PCR)
· PCR is artificial DNA replication can be carried out on tiny samples of NDA to generate multiple copies of the sample. This
is useful in forensic investigations where DNA samples have to be `amplified' to make enough material for genetic profiling.
· PCR can only replicate short sequences of DNA, not entire chromosomes like natural DNA replication.
PCR is a Cyclic Reaction
1. The DNA sample is mixed with DNA nucleotides + the enzyme DNA polymerase.
2. The mixture is heated to 95c this breaks the hydrogen bonds holding the strands together so the samples are now single-
3. Short lengths of single-stranded DNA are added primers.
4. The mixture is cooled to about 55c so the primers can bind + form small sections of double-stranded DNA at either end of the
5. The DNA polymerase can bind to these double-stranded sections.
6. The mixture is heated to 72c the enzyme extends the double-stranded section by adding free nucleotides to the unwound
7. When the DNA polymerase reaches the other end of the DNA strand, a new double-stranded DNA molecule is generated.
8. The whole process can be repeated many time so lots of DNA can be made.
9. Described as `thermophilic' because it is not denatured by the extreme temperatures used in this process.…read more
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2.2.12 Introduction to Genetic Engineering
· Genetic Engineering is a broad term used to describe a number of different processes for obtaining a specific gene and
placing that gene in another organism. The organism receiving the gene expresses the new gene product such organisms
are described as transgenic.
· The processes involved in genetic engineering are called recombinant DNA technology because they involve combining DNA
from different organisms or from different sources in a single organism.
· In genetic engineering the following steps are necessary:
1. The required gene is obtained.
2. A copy of the gene is placed in a vector.
3. The vector carries the gene to the recipient cell.
4. The recipient expresses the gene through protein synthesis.
· Restriction enzymes are used to cut through DNA at specific points.
· A particular restriction enzyme will cut DNA wherever a specific base sequence occurs + only where that sequence occurs.
This sequence is called the restriction site usually less than 10 base pairs.
· Restriction enzymes gives a `staggered cut' which leaves some exposed bases known as a sticky end.
· When separate fragments of DNA need to be stuck together, DNA ligase is used.
· In order to join together DNA fragments from different sources both need to have originally been cut with the same
restriction enzyme the sticky ends are complementary + allow bases to pair up.
· When DNA fragments from different organisms are joined in this way, the resulting DNA is called recombinant DNA.…read more