1. DNA Technology

2. DNA technology uses: Genetic engineering
Genetically modified (GM) crops
Gene Therapy
Genetic (DNA) Fingerprinting
Genetic Screening
Personalised medicine

3. Techniques used in Dna tech
Amplifying DNA: PCR
Comparing DNA: identifying and using specific marker sites using restriction endonuclease enzymes & DNA probes

4. Polymerase
Chain
Reaction

5. PCR is used in:
forensic science e.g. genetic fingerprinting;
medical research e.g. genetic screening to find alleles for genetic diseases;
scientific research e.g. to investigate DNA of extinct species to determine their relationship with living organisms;
The Human Genome Project.
Its purpose is to amplify very small quantities of DNA producing, many identical copies of the selected section of DNA in a very short time.

6. method
Heat to 95oC to break hydrogen bonds between complementary bases and separate strands
Cool to 40-60oC & add excess primer
Primers are short single stranded sequences of about 20 nucleotides which are complementary to bases in part of the DNA strand being copied.
Cooling allows the primers to bind (anneal) to each DNA strand at specific complementary points
Primers stop the DNA strands rejoining
They bracket the DNA to be copied by attaching to the end of the DNA sequence and shows DNA polymerase where to start copying. There are 2 different primers as there are 2 different ends of the DNA
DNA replication can only start with a double stranded region

7. Heat to 70oC. Add free DNA nucleotides & thermostable DNA polymerase
Polymerase copies each strand , starting with the primers.
The polymerase is thermostable allowing the process to occur rapidly at the higher temperatures required, without denaturing. The enzyme was originally isolated from a bacterium that lives in hot springs.
TWO DNA molecules will be formed following PCR and are used as templates as the process is repeated. This allows millions of copies of the original DNA to be produced in a very short time.

8. HEAT 95oC HEAT 70oC Cool 40-60oC primer DNA polymerase primer
Heat causes Hydrogen bonds between bases to break.
Heat acting in same way as DNA polymerase during replication.

9. key requirement AVOID CONTAMINATION with any other DNA source
as both the original & contaminated DNA will be amplified
Double Helix heated, unwinds 9

10. The Polymerase Chain Reaction sumanisinc
brill animation

11. that are used to identify specific DNA sequences
genetic markers
sections of DNA
that are used to identify
specific DNA sequences
Allows for comparison of DNA sequences between different people

12. restriction endonuclease enzymes
Enzymes that recognise & cut specific nucleotide sequences.
Originally from bacteria that use RE enzymes to cut DNA of attacking bacteriophages.

13. produces sticky ends because G & A don’t lie opposite each other.
e.g. EcoR1
recognises the sequence GAATTC, cutting between the G & A of each strand
produces sticky ends because G & A don’t lie opposite each other.

14. 3’ CCCTAGG 5’ GGGATCCTTAA 3’ CCCTAGGAATTCAGACC 5’ AATTCAGACC 5’
GTCTGG 3’
5’ GGGATCCTTAAGTCTGG 3’
sticky ends

15. e.g. Bam1
recognises the sequence GGATCC, cutting between the G & G of each strand
produces sticky ends because G & G don’t lie opposite each other.
…G G A T C C…
…C C T A G G…

16. e.g. Hae 111
recognises the sequence GGCC, cutting between the G & C of each strand
produces blunt ends because G & C lie opposite each other, these are not as useful as sticky ends.
…G G C C…
…C C G G…

17. Restriction enzymes will cut DNA into a number of fragments, depending upon the number of recognition sites in the sample of DNA.
The size of the fragment depends on how far apart the recognition sites are.
If DNA has 4 recognition sites, 5 fragment lengths of DNA are produced.
Recognition
site 1
Recognition
site 2
Recognition
site 3
Recognition
site 4
DNA SAMPLE
5 fragments

18. If two samples of DNA are identical, treatment with a particular restriction enzyme will produce the same number of DNA fragments, each of similar size.
However, different individuals have different DNA sequences and therefore produce different fragments when a particular restriction enzyme is used to cut an equivalent section of DNA.
The fragments produced are called RFLPs, RESTRICTION FRAGMENT LENGTH POLYMORPHISMS.

19. This allows for comparison or identification of DNA samples
PERSON A
Recognition
site 1
Recognition
site 2
Recognition
site 3
Recognition
site 4
DNA SAMPLE
5 fragments
PERSON B
Recognition
site 1
Recognition
site 2
Recognition
site 3
Recognition
site 4
Recognition
site 5
DNA SAMPLE
6 fragments
This allows for comparison or identification of DNA samples

20. Restriction Fragment Length Polymorphisms
RFLPS
pronounced RiFlips
Restriction Fragment Length Polymorphisms

21. reasons why different people
have different DNA sequences
SNPs
MRSs

22. Single Nucleotide Polymorphisms
snps
pronounced snips
Single Nucleotide Polymorphisms

23. A SNP is …… a difference in a single nucleotide in a section of DNA
It can give rise to different alleles for a gene
egs include sickle cell anaemia (chromosome 11)

24. Sickle cell anaemia Thr Pro Glu Glu Glu ...ACT CCT GAG GAG GAG... Thr
normal Hb chain
Amino acid sequence
Thr
Pro
Glu
Glu
Glu
DNA base sequence
...ACT
CCT
GAG
GAG
GAG...
codon number 4 5 6 7
sickle cell Hb chain
Amino acid
sequence
Thr
Pro
Val
Val
Glu
DNA base sequence
...ACT
CCT
GTG
GTG
GAG...

25. Chromosome 16 Genetically Ginger RSci Advent gcsbio.weebly.com

26. Microsatellite Repeat Sequences
MRSs
Microsatellite Repeat Sequences
(Also called variable number tandem repeats – VNTRs).

27. Only approximately 2% DNA codes for proteins and 3% has a regulatory role. The remaining 95% has unknown function and is called non-coding DNA.
Within this DNA there are many sections where a small number of bases are repeated many times
e.g. ….ATCATCATCATCATCATCATC…

28. These sequences are called microsatellite repeat sequences or short tandem repeats.
The number of repeats of these MRSs are unique to an individual.

29. Short sequences of single stranded DNA
dna PROBE
Short sequences of single stranded DNA
with known nucleotide and base sequence.
May be fluorescent or radioactively labelled, to enable it to be located.
Radioactive probes contain radioactive isotopes of nitrogen in their nucleotide structure.

30. The DNA is treated to separate the DNA strands (heat)
Add probe – it base pairs with complementary DNA sequence.
Detect the target sequence:
Fluorescent probe use a laser scanner
Radioactive probe use X-ray film

31. They are used to identify specific gene sequences .
Many inherited diseases are caused by a single allele with a known base sequences
A probe complementary to the allele nucleotide sequence is used:
If it binds the disease is present
If it doesn’t bind it is not present.

32. Pre implantation genetic diagnosis
PGD
Pre implantation genetic diagnosis
This is the use of DNA probes to detect defective genes in an embryo to reduce the chance of a hereditary disease in a family.

35. Measuring genetic variation allows the following questions to be answered:
Does a particular gene differ from person to person? Are two persons closely related?
Are certain alleles associated with an hereditary disease?
How do genes vary within a population? How much genetic diversity is there within a particular species?
How do genes differ from species to species? How can this be used to unravel the taxonomic relationship among species?

37. Genetic (dna) fingerprinting
Extract DNA from a sample
e.g. from a crime scene, often PCR required to amplify the DNA
2. Restriction Enzymes cut the DNA into different sized fragments
Specific MRSs used as they can be measured more accurately than RFLPs. Primers used to flank the MRS region involved to ensure fragments only come from that region

38. 3. DNA fragments separated using gel electrophoresis.
DNA is placed into separate wells in agar gel.
An electric current is applied across the length of the gel, with the negative electrode closest to the wells of DNA.
DNA has a negative charge, so moves through the spaces in the gel towards the positive electrode.
Smaller DNA fragments move further than larger fragments.
4. DNA is heated to separate the DNA strands to make it single stranded.

39. The gel will dry up, so the DNA fragments are transferred to a nylon membrane (process called Southern blotting)
Radioactive or fluorescent DNA probes are added to the membrane and will join to the complementary base sequences in the microsatellite region selected. The sample is washed to remove any probes that do not base pair.
The labelled DNA is added to X-ray film or a laser scanner is used to detect the radioactive or fluorescent probes and a pattern of bars appear, revealing the position of the DNA fragments: a DNA fingerprint.
Diagram p82

40. definitions
PROBES
GENETIC MARKERS
SNPS
RFLPS
MRSs