1. V) BIOTECHNOLOGY

2. DNA Sequencing
DNA sequencing determines the exact sequence of base pairs for a particular DNA fragment
the first method to sequences DNA was called chain terminating
it involved replicating DNA using chain terminating nucleotides that created small DNA fragments.
the fragments are then run on a gel matrix.

3. this method was developed by Fredrick Sanger, Alan Maxim and Walter Gilbert
variations of this method was used by the human genome project.

5. The Human Genome Project
Started in 1990 and finished in 2008 (James Watson!)
Estimates 3 billion base pairs of nucleotides.
The haploid human genome contains ~23,000 protein-coding genes, far fewer than had been expected before its sequencing. In fact, only about 1.5% of the genome codes for proteins, while the rest consists of non-coding RNA genes, regulatory sequences, introns, and (controversially named) "junk” DNA.
Aim of the project: new drugs and genetic therapies to combat genetic diseases such as CF, Huntington, etc
Earlier predictions estimated that human cells have as much as 200,000 genes

6. DNA Sequencing Recombinant DNA
fragment of DNA composed of sequences originating from at least two different sources.
Genetic Transformation
introduction and expression of foreign DNA in a living organism.

8. Tools of Biotechnology
Restriction Endonucleases
also called restriction enzymes
act like molecular scissors cutting double-stranded DNA at a specific base pair sequence
each type of restriction enzyme recognizes a particular sequence of nucleotides.
this is known as the recognition site.

9. molecular biologists use these enzymes to cut DNA in a predictable and precise way.
most recognition sites are four to eight base pairs long and are usually characterized by a complementary palindromic sequence.
example EcoR1
Enzyme Source Recognition Sequence Cut
EcoR1 Escherichia coli 5'GAATTC 5’…G AATTC…3'
3'CTTAAG 3’…CTTAA G…5'

10. EcoR1 scans the DNA molecule and binds at its recognition site.
once bound it cuts between the Adenine and Guanine nucleotide on each strand
at the end of each cleavage site one strand is longer than the other
it has exposed base pairs that lack complementary base pairs.
these are called sticky ends.

12. not all restiction enzymes produce sticky ends.
Enzyme Source Recognition Sequence Cut
Sma1 Serratia marcescens 'CCCGGG 5'---CCC GGG---3' 'GGGCCC '---GGG CCC---5'
the ends of the DNA fragments are fully base paired.
these ends are called blunt ends.
blunt ends have no sticky ends.

13. restriction enzymes that produce sticky ends are generally more useful as a biological tool.
sticky ends can be joined to other DNA fragments created by the same restriction endonuclease
molecular biologists select restriction endonucleases that cut around genes of interest
restriction enzymes are named according to the bacteria they come from.
first letter is the genus name
second and third letter are the initial letters of the species name.
fourth letter indicates the strain
numbers represent the order of discovery.

14. how many fragments will be produced is Sma1 digests this sequence? 3
5'-AATTCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTAGCA-3'
3'-TTAAGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAATCGT-5‘
how many fragments will be produced is Sma1 digests this sequence? 3
What types of ends does Sma1 produce?
Blunt

15. Methylases
enzymes that can modify a restriction enzyme site
adds a methyl (-CH3) group to one of the bases in the site
they protect a gene fragment from being cut at an undesired location
In bacteria, it’s a crude type of immune system that was observed to prevent E.coli from being infected by viruses.
In eukaryotes, usually occurs to inactivate a specific gene.

16. Ligases
to create recombinant DNA, pieces of DNA from two sources must be joined together.
using restriction enzymes and methylases, geneticists can engineer fragments of DNA with desired genes
these fragments are joined together by DNA ligase.
DNA ligase joins sticky ends together.

21. Taq DNA Polymerase and the Polymerase Chain Reaction
in 1985 Kary Mullis invented the technique called the polymerase chain reaction (PCR)
PCR allows scientists to make billions of copies of pieces of DNA from extremely small quantities.
the reaction depends on the Taq DNA polymerase
Taq DNA polymerase synthesises DNA during replication
works at very high temperatures because the bacteria it comes from lives in high temperatures (thermophile)
each cycle of PCR doubles the number of DNA molecules.

22. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8
TECHNIQUE 5 3
Target sequence
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Figure 20.8 The polymerase chain reaction (PCR)
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

23. TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a
Figure 20.8 The polymerase chain reaction (PCR)

24. Cycle 1 yields 2 molecules
Fig. 20-8b 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
Figure 20.8 The polymerase chain reaction (PCR)
New nucleo- tides

25. Cycle 2 yields 4 molecules
Fig. 20-8c
Cycle 2 yields 4
molecules
Figure 20.8 The polymerase chain reaction (PCR)

26. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8d
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence
Figure 20.8 The polymerase chain reaction (PCR)

27. Transformation of Bacteria
Genes are isolated and then inserted into a plasmid.
Plasmids are circular double stranded DNA that occur naturally in many bacteria.
Homework: Read pages of your textbook and then answer question 8, due tomorrow.