1. studying genomes

2. Studying DNA
- all following techniques are basic tools of biotechnology (genetic engineering)

3. Enzymes for DNA manipulation
Before 1970s, the only way in which individual genes could be studied was by classical genetics.
Biochemical research provided (in the early 70s) molecular biologists with enzymes that could be used to manipulate DNA molecules in the test tube.
Molecular biologists adopted these enzymes as tools for manipulating DNA molecules in pre-determined ways, using them to make copies of DNA molecules, to cut DNA molecules into shorter fragments, and to join them together again in combinations that do not exist in nature.
These manipulations form the basis of recombinant DNA technology.

4. Recombinant DNA technology
The enzymes available to the molecular biologist fall into four broad categories:
DNA polymerase – synthesis of new polynucleotides complementary to an existing DNA or RNA template
Nucleases – degrade DNA molecules by breaking the phosphodiester bonds
restriction endonucleases (restriction enzyme) – cleave DNA molecules only when specific DNA sequences is encountered
Ligases – join DNA molecules together
End modification enzymes – make changes to the ends of DNA molecules

5. endonucleases - make cuts at internal phosphodiester bonds
exonucleases - remove nucleotides from the ends of DNA and/or RNA molecules
endonucleases - make cuts at internal phosphodiester bonds
source: Brown T. A. , Genomes. 2nd ed.

6. DNA cloning
DNA cloning (i.e. copying) – logical extension of the ability to manipulate DNA molecules with restriction endonucleases and ligases
vector
DNA sequence that naturally replicates inside bacteria.
It consists of an insert (transgene) and larger sequence serving as the backbone of the vector.
Used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes.
expression vector (expression construct) - generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable mRNA, and therefore proteins. Expression vectors require sequences that encode for polyadenylation tail, minimal UTR sequence, Kozak sequence.

7. Vectors
plasmid
DNA molecule that is separated from, and can replicate independently of, the chromosomal DNA.
Double stranded, usually circular, occurs naturally in bacteria.
Serves as an important tool in genetics and biotechnology labs, where it is commonly used to multiply (clone) or express particular genes.
length of insert: 1-10 kbp
source: wikipedia

8. Vectors BAC (bacterial artificial chromosome) cosmid
It is a particular plasmid found in E. coli. A typical BAC can carry about 250 kbp ( kbp).
cosmid
40-45 kbp
YAC (yeast artificial chromosome)
Mbp
YAC is an eukaryotic plasmid

9. restriction endonuclease
ligase
An animal gene has been obtained as a single restriction fragment after digestion of a larger molecule with the restriction enzyme BamHI.
Small E. coli plasmid has been purified and treated with BamHI, which cuts the plasmid in a single position.
The circular plasmid has therefore been converted into a linear molecule. Mix the two DNA molecules together and add DNA ligase.
Various recombinant ligation products will be obtained, one of which comprises the circularized plasmid with the animal gene inserted into the position originally taken by the BamHI restriction site.
If the recombinant plasmid is now re-introduced into E. coli, and the inserted gene has not disrupted its replicative ability, then the plasmid plus inserted gene will be replicated and copies passed to the daughter bacteria after cell division. More rounds of plasmid replication and cell division will result in a colony of recombinant E. coli bacteria, each bacterium containing multiple copies of the animal gene.
This series of events constitutes the process called DNA or gene cloning.
DNA cloning
source: Brown T. A. , Genomes. 2nd ed.

10. PCR – Polymerase chain reaction
DNA cloning results in the purification of a single fragment of DNA from a complex mixture of DNA molecules.
Major disadvantage: it is time-consuming (several days to produce recombinants) and, in parts, difficult procedure.
The next major technical breakthrough (1983) after gene cloning was PCR.
It achieves the amplifying of a short fragment of a DNA molecule in a much shorter time, just a few hours.
PCR is complementary to, not a replacement for, cloning because it has its own limitations: we need to know the sequence of at least part of the fragment.
PCR –see more at
Unlike cloning, PCR is a test-tube reaction and does not involve the use of living cells: the copying is carried out not by cellular enzymes but by the purified, thermostable DNA polymerase of T. aquaticus.

11. Mapping genomes

12. What is it about?
Assigning/locating the specific gene to the particular region at the chromosome and determining the location and relative distances between genes at the chromosome.
There are two types of maps:
genetic linkage map – shows the arrangement of genes (or other markers) along the chromosomes as calculated by the frequency with which they are inherited together
physical map – representation of the chromosomes, providing the physical distance between landmarks on the chromosome, ideally measured in nucleotide bases
The ultimate physical map is the complete sequence itself.
Cerpano z kapitoly Chapter 5 - Mapping Genomes z knihy Genomes,
A genetic map provides an indirect estimate of the distance between two items and is limited to ordering certain items.
Genetic map is like an interstate highway map, it serves to guide a scientist toward a gene, just like an interstate map guides a driver from city to city.
On the other hand, physical maps mark an estimate of the true distance, in measurements base pairs, between items of interest.
To continue our analogy, physical maps would then be similar to street maps, where the distance between two sites of interest may be defined more precisely in terms of city blocks or street addresses.
The different types of maps vary in their degree of resolution, that is, the ability to measure the separation of elements that are close together. The higher the resolution, the better the picture.

13. Genetic mapping
use of genetic techniques to construct maps showing the positions of genes
genetic techniques:
cross-breeding experiments
humans: the examination of family histories (pedigrees)

14. Genetic linkage map Constructed by observing how frequently
two markers (e.g. genes) are inherited together.
Two markers located on the same chromosome can be separated only through the process of recombination.
If they are separated, childs will have just one marker from the pair.
However, the closer the markers are, the more tightly linked they are, and the less likely recombination will separate them. They will tend to be passed together from parent to child.
Recombination frequency provides an estimate of the distance between two markers.
Genes A and B are on the same chromosome and so should be inherited together. A a B jsou linked, dedi se dohromady. Jediny zpusob, jak se odseparuji, je v prubehu rekombinace. Cim jsou ale blize, tim je sance separace nizsi.
A a C nebo B a C nejsou linked, dedi se nezavisle. Tohle pozoroval Mendel, Mendeluv druhy zakon rika, ze pary alel se dedi nezavisle. Mendel neobjevil linkage, protoze 7 genu, ktere studoval, byly kazdy na jinem chromozomu.
Genomes 2, T. A. Brown

15. Look at the results of meiosis in a hundred identical cells.
If crossovers never occur then the resulting gametes will have the following genotypes:
200 AB, 200 ab
This is called completed linkage.
But if (as is more likely) crossovers occur between A and B in some of the nuclei, then the allele pairs will not be inherited as single units. Let us say that crossovers occur during 40 of the 100 meioses. The following gametes will result:
160 AB, 160 ab, 40 aB, 40 aB
This is called partial linkage.
The recombination frequency depends on the distance between two genes.
The drawing shows a pair of homologous chromosomes, one red and the other blue. A and B are linked genes with alleles A, a, B and b. On the left is a meiosis with no crossover between A and B: two of the resulting gametes have the genotype AB and the other two are ab. On the right, a crossover occurs between A and B: the four gametes display all of the possible genotypes: AB, aB, Ab and ab.
predpoklady
rekombinace se objevuje nahodne (ale neni to uplne pravda, existuji recombinant hotspots)
vztah vzdalenost/frekvence je linearni
Poruseni techto predpokladu vede k tomu, ze geneticke mapy jsou mene presne.
Genomes 2, T. A. Brown

16. Genetic markers
A genetic map must show the positions of distinctive features – markers.
Any inherited characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker.
Markers can be
genes
DNA segments that have no known coding function but which inheritance pattern can be followed.
- markers are recognizable components of the landscape, such as rivers, roads and buildings

17. Genetic linkage map
On the genetic maps distances between markers are measured in terms of centimorgans (cM).
1cM apart – they are separated by recombination 1% of the time
1 cM is ROUGHLY equal to physical distance of 1 Mbp in human
named after American geneticist Thomas Hunt Morgan
Just a bit more about marker analysis:

18. Genes as markers useful but no ideal
For larger genomes (e.g. vertebrates), gene maps are not very detailed (genes are widely spaced out with large gaps between them).
Variations within genes lead to observable changes (e.g. eye color). However, only a fraction of the total number of genes exist in allelic forms that can be distinguished conveniently.
Gene maps are therefore not very comprehensive. We need other types of marker.
Mapped features that are not genes are called DNA markers.

19. DNA markers
Must be polymorphic, i.e. alternative forms (alleles) must exist among individuals so that they are detectable among different members in family studies.
Most variations occur within introns, have little or no effect on an organism, yet they are detectable at the DNA level and can be used as markers.
restriction fragment length polymorphisms (RFLPs)
simple sequence length polymorphisms (SSLPs)
single nucleotide polymorphisms (SNPs, pronounce “snips”)

20. RFLPs (restriction fragment length)
Recall that restriction enzymes cut DNA molecules at specific recognition sequences.
This sequence specificity means that treatment of a DNA molecule with a restriction enzyme should always produce the same set of fragments.
This is not always the case with genomic DNA molecules
because some restriction sites exist as two alleles, one allele
displaying the correct
sequence for the restriction
site and therefore being cut,
and the second allele having
a sequence alteration so the
restriction site is no longer
recognized.
source: Brown T. A. , Genomes. 2nd ed.

21. SSLPs (simple sequence length)
Repeat sequences that display length variations, different alleles contain different numbers of repeat units (i.e. SSLPSs are multi-allelic).
variable number of tandem repeat sequences (VNTRs, minisatellites)
repeat unit up to 25 bp in length
simple tandem repeats (STRs, microsatellites)
repeats are shorter, usually di- or tetranucleotide
source: Brown T. A. , Genomes. 2nd ed.

22. SNPs (sigle nucleotide)
Positions in a genome where some individuals have one nucleotide and others have a different nucleotide.
Vast number of SNPs in every genome.
Each SNP could have potentially four alleles, most exist in just two forms.
The value of two-allelic marker (SNP, RFLP) is limited by the high possibility that the marker shows no variability among the members of a family.
The advantages of SNP over RFLP:
they are abundant (human genome: 1.5 millions of SNPs, RFLPs)
easire to type (i.e. easier to detect)

23. Marker analysis Value of genetic map – marker analysis
Inherited disease can be located on the map by following the inheritance of a DNA marker present in affected individuals (but absent in unaffected individuals), even though the molecular basis of the disease may not yet be understood nor the responsible gene identified.
This represent a cornerstone of testing for genetic diseases.

24. Physical maps
A map generated by genetic techniques is rarely sufficient for directing the sequencing phase of a genome project.
The resolution of a genetic map depends on the number of crossovers that have been scored.
Genetic maps have limited accuracy.
Before large-scale sequencing begins (see next lecture), a genetic map must be checked and supplemented by alternative mapping procedure.
ad 1. – neni problem pro bakterie, kde mam k dispozici mnoho jedincu, pro lidi je ale treba problem, ze se da studovat relativne malo meioz
ad 2. - assumption that crossovers occur at random along chromosomes is only partly correct because the presence of recombination hotspots means that crossovers are more likely to occur at some points rather than at others.

25. Physical mapping
A plethora of physical mapping techniques has been developed to address this problem, the most important being
Restriction mapping
Fluorescent in situ hybridization (FISH)
Sequence tagged site (STS) mapping
see more at Genomes2, Brown,

26. Genetic and physical map - compared
Saccharomyces cerevisiae chromosome III
physical map obtained by DNA sequencing
the order of the upper two markers (glk1 and cha1) is incorrect on the genetic map
there are also differences in the relative positioning of other pairs of markers
Oliver SG et al., Nature, 357, 38–46

27. the order and spacing of the markers, measured in base pairs
more at
Genome maps
relative locations of markers are established by following inheritance patterns
visual appearance of a chromosome when stained and examined under a microscope
the order and spacing of the markers, measured in base pairs
genetic map – cM … centimorgan, The higher the percentage of recombinants for a pair of traits, the greater the distance separating the two loci. In fact, the percent of recombinants is arbitrarily chosen as the distance in centimorgans (cM). see
Chromosome mapping by counting recombinant phenotypes produces a genetic map of the chromosome.
As a very rough rule of thumb, 1 cM on a chromosome encompasses 1 megabase of DNA.
The lowest-resolution physical map is the chromosomal or cytogenetic map, which is based on the distinctive banding patterns observed by light microscopy of stained chromosomes.
sequence map
source: Talking glossary of genetic terms,

28. NCBI human Genome Resources