Genetic Engineering

Genetic Engineering

The Objective : To give information about : 1- What genetic engineering means and how DNA segments can be used in vitro . 2- Types of genetic techniques used in genetic engineering : * Recombinant DNA * DNA cloning * Genetic fingerprint * Polymerase chain reaction ( PCR ) 3- Application of genetic engineering 4- How genetic technology is used in the diagnosis of genetic diseases and in forensic medicine and crime detection . 5- How DNA is used to determine the human genetic identity . 6- How DNA segments can be quickly and efficiently amplified millions of times using PCR . 7- How genetic technology involved genetically modified organisms and their products in a wide range of activities involving medicine , agriculture , and industry .

Genetic Engineering

Genetic Engineering : also called genetic modification is the direct human manipulation of an organisms genetic material in away that does not occur under normal conditions . Any organism that is generated using these techniques is considered to be a genetically modified organism .


Recombinant DNA technology : Adds genes from one type of organism to the genome of another . Constricting Recombinant DNA : Manufacturing Recombinant DNA require Restriction enzymesRestriction Endonuclease : cut DNA at specific nucleotide sequence called restriction site . they recognize DNA sequences that are Palindromes (sequences read the same at either direction) ( Figure 1) 5 – GAATTC – 33 – CTTAAG – 5


Cleavage of the DNA at this site produces complementary single stranded tails ( called sticky ends) . These single stranded tails can annealed with single stranded tails from other DNA fragments by Ligase enzymes that after ligation form Recombinant DNA . ( Figure 2 ) There are 2 types of restriction enzymes depending on cleavage product producing sticky ends or blunt ends

Cloning DNA :

specific DNA segments are inserted into vectors (such as Plasmids) which are transferred into host cells (such as bacterial cells) where the recombinant molecules replicate as the host cells divide . Steps in Cloning DNA Techniques : The DNA to be cloned is isolated and treated with restriction enzyme to creat sticky ends . The fragments are then linked to plasmid molecules that have been cut with the same restrictin enzyme creating a collection of recombinant vectors . The recombinant vectors are transferred into E. coli host cells . The bacterial cell is grown in culture to increase numbers . cells are induced to transcribe and translate the foreign gene Synthesis yields a protein product (such as insulin) that is recovered from the culture and purified . Human insulin produced by E.coli was one of first genetically engineered proteins to be commerically produced (figure 3 ) . Plasmids : are extrachromosomal self replicating circular form of DNA present in most bacteria .

Application of Cloning DNA Technique :

1- Application of genetic engineering has an importance in medicine, agriculture , and industry . It main uses are : 2- Production of large number of piece of DNA carrying a gene for efficient production of useful proteins such as : Human insulin , enzymes , drugs , hormones , vaccines , and blood clotting factor lacking in person with hemophilia 3- Creation of cells capable of synthesizing economically important molecules such as antibiotics . 4- Generation of DNA and RNA sequences as research tools or in medical diagnosis . 5- Manipulation of genotype of organism such as plants . plants are engineered for drought, insect , and virus resistance . 6- Potential correction of genetic defects in animals including human patient ( gene therapy ) .


Genetic Fingerprint :
( also called DNA profile , DNA typing , DNA testing , DNA fingerprinting ) : is atechnique used to distinguish between individuals of the same species using only samples of their DNA . It determines the pattern of certain parts of genetic material DNA that is unique to each individual ( except identical twins ) . used to rule out establish identity . Discovery and Principles of Genetic Fingerprint : The beginnings of DNA profiling can be traced in the work of Britain geneticist Alec Jeffreys in (1985) . He found that the myoglobin gene contains many segments that vary in size and composition from individual to individual and that have no apparent function . Jeffreys called these segments minisatellites. DNA variation is the substrate of genetic fingerprint . 99.7 % of human DNA is identical between individuals but the 0.3% that differs enables scientists to distinguish identity . DNA profiling based on the analysis of minisatellite (VNTRs) or microsatellite (STRs) .

* DNA profiling based on DNA minisatellites or Variable Numbers of Tandem Repeats (VNTRs) : (VNTR) : repeating units of DNA sequences consist of (10-100 nucleotide) which number varies between individual . (Figure 4) Asignificant limitation of VNTR profiling is that requires arelatively large sample of DNA more than is usually found in atypical crime scene and the DNA most be relatively intact (not degraded ) as aresult VNTR profiling is used most frequently when large tissue samples are available such as paternity test .


Schematic of minisatellite and microsatellite (STR) DNA markers. PCR primers are designed to target invariant flanking sequence regions. The number of tandem repeat units in the repeat regions varies among individuals making them useful markers for human identification.

* DNA profiling based on DNA microsatellites or Short Tandem Repeats (STRs) : Short DNA sequences consist of (2-9 bases long) that are repeated in tandem . These repeated sequences often exist in a chromosomal region called heterochromatin alargely unused portion of DNA found in each chromosome . ( Figure 5 )

The number of repeats is present in agiven STR locus often varies between individual and between population . The FBI have selected 13 STR loci as to be used a core set for forensic analysis . DNA profiles based on these loci are stored in a national DNA database called Combined DNA Index System ( CODIS ) . as in ( figure 6 ) .
D: DNA 7: chromosome 7 S: single copy sequence 820: 820th locus described on chromosome 7
CODIS thirteen STR sequences that have been selected for the Combined DNA Index System(CODIS)


The major advantage of STR profiling is that : 1 - they can be generated from trace samples (eg single hairs or saliva left on acigarette butt ) 2 - The small size of these repeats makes them more likely to persist in old or degraded DNA (eg skull found in a field or ancient Egyptian mummy ) . 3 - STR are easily amplified by PCR so it is rapidly replacing VNTR typing in many profiling laboratories .


Genetic Marker System (Source of Genetic Identity) 1- STR found in the Intron region of Autosomal chromosomes (nuclear chromosomes ). ( Figure 7 )



2- STR in the Sex chromosome Y- chromosome : a- Y chromosome are paternally inherited so Y- STR analysis can help in the identification of paternally related males . b- Y – STR used in Rape cases . 3- STR found in the Mitochondrial DNA (mt DNA) : a- If nuclear DNA is extremely damaged two regions of repeats in mt DNA that are highly variable in population may yield information . ( figure 8 )

b- Because mtDNA is maternally inherited directly linked maternal relatives can be used as match references such as ones maternal grandmothers , sisters son . C- Mt DNA can be obtained from such materials hair shafts and old bones and teeth .

Mitochondrial DNA

Sample collection and labeling
DNA extraction
DNA amplification reaction(PCR)
DNA separation and detection
(electrophoresis, genetic analyzer)
Data Analysis Steps
Genetic finger print steps process
Data Review and evaluation
(Lisa mobile)
Methods of Genetic fingerprint

Biological Sample

Personal Items

Methods of Genetic fingerprint :

1- Sample Collection : Sample collected to obtain DNA from : blood , bone, teeth , tissue , biopsy , urine , skin , saliva , hair , nail, also buccal swab . also sample collected from personal items (eg tooth brush , hair brush , cup , razor ) .

2- DNA Extraction and Purification : There are 3 techniques for DNA extraction

1- organic extraction ( Phenol Chloroform extraction ) 2. chelex extraction 3- FTA paper extraction is an absorbent cellulose –based paper .

Alec Jeffreys detected differences in numbers of VNTR or STR among individuals by cutting these regions of DNA with restriction enzymes . 3- DNA amplification using Polymerase Chain Reaction (PCR ) : STR amplify using PCR PCR : Technique allows researchers to amplify atiny sample of DNA millions of times in few hours in vitro. PCR Principles and Procedure : PCR is used to amplify specific regions of a DNA strand ( the DNA target ) . This can be a single gene , a part of a gene , or a non coding sequence There are 3 major steps in PCR which are repeated for 30 or 40 cycles.

PCR


PCR Steps:A- Denaturation step at 95 C : It causes melting of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.(Figure 12) B– Primer Annealing step at 54 C : Annealing of the primers to the single stranded DNA template . C–Extension or Elongation step at 72 C: DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding deoxynucleotide triphosphate (dNTPs) that are complementary to the template in 5 to 3 direction .


4 – DNA Separation using Gel Electrophoresis or Capillary Gel Electrophoresis : The products of PCR ( DNA containing solutions ) are added to the wells of a special agar gel (agarose) and subjected to an electric current . Follow of electricity from negative pole to the positive pole causes DNA pieces (carrying negative charges because of their phosphate groups) to migrate in the gel substance twoard the positive pole . the rate of movement is based on the size of fragmenrs the smaller fragments migrate faster and are positioned closer to the bottom whereas the larger fragments move more slowly and remain nearer the top of the gel .( Figure 13 , 14 )

Agarose gel electrophoresis

DNA separation and detection



To determine the positions of DNA fragments the gel is stained with ethidium bromide and illuminating it with ultraviolet light . The various sized DNA fragments appears as a serious of bands . ( Figure 15 )

The pattern is compared against known standards . (Figure 16 ,17 ,18 ,19 , 20 , 21, 22)

Father
Child
Mother


Capillary Gel Electrophoresis : The amplified mixture is electrophoresed through a thin capillary gel tube (Figure 23 ) . Larger DNA fragments will be retarded in the capillary gel and smaller ones will pass through more quickly . At the bottom of the tube, a laser detects when each fluorescent DNA fragment exits the capillary tube and computer software assigns a size to the fragment . All fragments are represented as Peaks on a graph (figure 24) .

Internal Lan Standard (ILS)

Data Analysis Steps
5 – Data Interpretation : The DNA profile is then compared against another sample to determine whether there is a genetic match .


Application of PCR and Genetic fingerprint : 1 – Forensic Investigation : identification of suspect based on crime scene samples .2 – Paternity test3 – To identify victims in disaster where large numbers of people died at once such as in airplane crashes , large fires or military confliets.4 – Diagnosis of genetic diseases ( Mutation )5 – Microbiology : bacteria and virus identification .6 - genetic diversity ( similarity , distance )7 – Gene mapping ( human genome project )8 – Identify Pedigree , anthropology , archeology , neurobiology9 – Medical Diagnosis10 – DNA sequencing